ANP32A Human
Description
Molecular Structure and Domains
ANP32A is a 28 kDa protein characterized by two key domains:
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Leucine-Rich Repeat (LRR) Domain: Facilitates protein-protein interactions, particularly with influenza polymerase subunits .
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Intrinsically Disordered Acidic Region (LCAR): Rich in aspartic/glutamic acids, enabling electrostatic interactions with basic surfaces of viral proteins .
Critical Viral Adaptations Enabled by ANP32A
| Mutation | Functional Impact | Associated ANP32A Interaction |
|---|---|---|
| PB2-E627K | Restores polymerase activity in mammals | Enhances LCAR binding affinity |
| PB2-D701N | Compensates for ANP32A incompatibility | Alters LRR domain interaction |
| PB2-Q591R | Facilitates mammalian ANP32A/B utilization | Modulates electrostatic interface |
Redundancy with ANP32B
Human ANP32A and ANP32B are functionally redundant but exhibit isoform preferences:
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ANP32B supports avian-derived polymerases more efficiently than ANP32A .
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SUMOylation at K68/K153 (ANP32A) or K68/K116 (ANP32B) enables NS2 protein recruitment during avian influenza adaptation .
Chromatin Remodeling
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Acts as a histone chaperone, facilitating nucleosome assembly/disassembly .
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Modulates transcriptional regulation via interactions with SET/TAF1A .
Apoptosis Regulation
Cancer Progression
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Promotes hepatocellular carcinoma proliferation via HMGA1/STAT3 pathway activation .
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Overexpression linked to poor prognosis in breast and pancreatic cancers .
Neurodegenerative Diseases
Recent Advances (2023–2024)
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SUMOylation-Dependent Viral Adaptation
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ANP32E Utilization
Research Tools and Antibodies
Product Specs
Q&A
What experimental systems are optimal for studying ANP32A’s role in influenza replication?
Methodological Answer:
Three primary approaches dominate current research:
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CRISPR-Cas9 knockout/rescue models: Dual ANP32A/B knockout HEK293T or eHAP1 cells are used to eliminate functional redundancy. Viral polymerase activity is restored via exogenous ANP32A transfection, quantified using minigenome assays (firefly luciferase reporter under viral promoter control) .
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In vitro reconstitution: Purified ANP32A IDD (residues 1–280) is combined with PB2 627-NLS domains (avian vs. human-adapted E627K mutants) for NMR or ITC binding assays. This identifies species-specific interaction motifs .
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Structural virology: Cryo-ET of influenza polymerase complexes in complex with ANP32A reveals spatial colocalization near the RNA exit channel, validated via mutagenesis (e.g., E178A/D179A in ANP32A disrupts NP binding) .
Table 1: Key ANP32A Domains and Functional Assays
| Domain | Functional Role | Assay Type | Readout Metrics |
|---|---|---|---|
| LRR (1–160) | Structural stabilization | X-ray crystallography | Binding affinity (Kd) |
| IDD (161–280) | Multivalent viral protein recruitment | NMR titration | Chemical shift perturbation |
| SIM (240–250) | SUMOylation-dependent NS2 interaction | Co-IP + Western blot | SUMO1/2/3 conjugation levels |
How does ANP32A deficiency affect influenza polymerase activity?
Methodological Answer:
Dual ANP32A/B knockout reduces viral RNA synthesis by >90% (measured via qRT-PCR of vRNA/cRNA) . Residual activity in single knockouts demonstrates functional redundancy:
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Human cells: ANP32A-/- + ANP32B-/- reduces IAV/IBV replication to undetectable levels (TCID50 < 10^1 PFU/mL) .
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Avian-human chimeras: Avian ANP32A packaged into virions (via PB1-PB2 binding) enhances early replication in human cells by 3-log units, bypassing adaptation requirements .
Critical Controls:
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Use ANP32A/B siRNA + rescue with siRNA-resistant plasmids to exclude off-target effects.
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Validate knockout efficiency via Western blot (anti-ANP32A antibody, Abcam ab229908) and RNA-FISH for viral genomes.
How do conflicting reports on ANP32A’s role in avian vs. human polymerase adaptation align?
Data Contradiction Analysis:
Early studies suggested strict species specificity (e.g., avian ANP32A supports avian polymerase only) , but recent findings show:
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Avian polymerase can use human ANP32A/B for cRNA synthesis (Step 1 replication) without adaptation .
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vRNA synthesis (Step 2) requires adaptive mutations (PB2-E627K) to engage human ANP32A’s truncated IDD .
Resolution Strategy:
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Step-specific assays: Separate cRNA (priming) and vRNA (elongation) synthesis using time-resolved radiolabeling (³²P-UTP incorporation).
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Single-molecule imaging: TIRF microscopy of FluPol-ANP32A complexes shows avian polymerase stalls at elongation without PB2-E627K .
What biophysical techniques resolve ANP32A’s dynamic interactions with viral proteins?
Methodological Answer:
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NMR relaxation dispersion: Quantifies µs-ms timescale dynamics in ANP32A IDD when bound to PB2-NLS or NP. Key parameters:
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Multi-angle light scattering (MALS): Confirms 1:1 stoichiometry for ANP32A-PB2 complexes but 2:1 for ANP32A-NP aggregates .
Case Study:
Deletion of the avian-specific 33-residue insertion (Δ169–201 in human ANP32A) reduces PB2-E627 binding affinity by 10-fold (Kd from 0.5 µM to 5 µM) . Restoration via chimeric constructs (e.g., huANP32A + avian 33-mer) rescues polymerase activity to 80% of avian levels .
How do post-translational modifications (SUMOylation) regulate ANP32A function?
Mechanistic Insights:
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SUMO E3 ligase PIAS2α mediates K68/K153 SUMOylation in ANP32A, creating a docking site for NS2’s SIM domain .
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Functional consequence: SUMOylated ANP32A recruits NS2 to enhance PB2/PA nuclear import, increasing polymerase assembly efficiency by 3.5-fold .
Experimental Validation:
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FRET-based SUMOylation assay: HeLa cells co-transfected with ANP32A-Flag, PIAS2α-HA, and SUMO1-GFP show FRET efficiency >25% in the nucleus .
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SENP1 inhibition: Treating cells with 50 µM SUMO protease inhibitor N-ethylmaleimide boosts ANP32A-NS2 colocalization (Pearson’s r = 0.82 vs. 0.32 in controls) .
Why do some studies report ANP32A-independent influenza replication?
Critical Analysis:
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Strain-specific effects: H3N2 (human-adapted) polymerases show partial ANP32A independence (40% residual activity in knockouts) versus H5N1 (<5%) .
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Alternative host factors: Subfunctionalization by ANP32E or ANP32D may compensate in certain cell types (e.g., A549 vs. HEK293T) .
Recommendations:
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Screen multiple influenza strains (≥3 subtypes) in parallel.
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Use isogenic PB2-E627K vs. wild-type viruses to isolate ANP32A dependency.
What limitations exist in current ANP32A interaction models?
Identified Issues:
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Overexpression artifacts: Endogenous ANP32A levels (≈2,000 copies/cell) are 10-fold lower than transfection-based studies .
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Lack of post-translational context: Most in vitro studies use unmodified ANP32A, ignoring SUMOylation/phosphorylation states .
Innovative Solutions:
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Endogenous tagging: CRISPR knock-in of HaloTag-ANP32A enables single-molecule tracking in live cells.
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Native mass spectrometry: Identifies stoichiometry of ANP32A-PB2-NP complexes under near-physiological conditions.
Product Science Overview
ANP32A is involved in several critical cellular processes, including:
- Tumor Suppression: ANP32A has been shown to promote apoptosis by favoring the activation of caspase-9 and allowing apoptosome formation .
- Regulation of Transcription: It plays a role in the modulation of histone acetylation and transcription as part of the INHAT (inhibitor of histone acetyltransferases) complex .
- Nucleocytoplasmic Transport: ANP32A is involved in the transport of molecules between the nucleus and the cytoplasm .
- RNA Binding: It has RNA binding activity, which is crucial for its role in regulating mRNA stability and processing .
The recombinant form of ANP32A is used in research to study its functions and interactions with other proteins. It is also a target for developing therapeutic interventions for diseases associated with its dysregulation.
