P58IPK Antibody

What is P58IPK and what is its role in viral infections?

P58IPK is a cellular protein that functions as an inhibitor of protein kinase R (PKR), an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2α. During viral infections, particularly influenza virus, P58IPK plays a crucial role in regulating viral mRNA translation. P58IPK is post-translationally activated during influenza virus infection and serves to combat the PKR antiviral response . It inhibits PKR dimerization, which would otherwise lead to translational shutdown and establishment of an antiviral state in infected cells . P58IPK represents what researchers have termed a "cellular inhibitor of the host defense" (CIHD), as it is activated during virus infection to inhibit virus-induced apoptosis and inflammation, prolonging host survival while simultaneously facilitating viral replication .

How does P58IPK function at the molecular level?

At the molecular level, P58IPK normally exists in an inactive complex with its negative regulator Hsp40. During viral infection, viral proteins like influenza nucleocapsid protein (NP) can bind to P58IPK with higher affinity than Hsp40, releasing P58IPK from this inhibitory complex. The NP-P58IPK complex is then selectively recruited to the 40S ribosomal subunit through direct interaction between NP and the ribosomal protein S19 (RPS19) . This positioning allows P58IPK to inhibit PKR, which is strategically located on the 40S ribosomal subunit where it has access to its downstream target eIF2α . The molecular mechanism involves NP serving as a bridge between P58IPK and the 40S ribosomal subunit, as NP has distinct binding sites for both P58IPK and RPS19 .

What are the common synonyms and identifiers for P58IPK?

P58IPK is encoded by the DNAJC3 gene in humans and is also known by several other names:

• DnaJ heat shock protein family (Hsp40) member C3

• ACPHD

• ERdj6

• HP58

This 504-amino acid residue protein has a molecular weight of approximately 58 kDa, which is reflected in its name and can be observed in Western blot applications . P58IPK is widely expressed across tissues, with particularly high expression levels in the pancreas and testis .

How are P58IPK antibodies used in viral infection studies?

P58IPK antibodies are essential tools for studying the dynamics of viral infections, particularly for investigating the PKR pathway inhibition. In experimental setups, these antibodies can be used to:

• Detect P58IPK activation status during different stages of viral infection

• Monitor P58IPK-viral protein interactions through co-immunoprecipitation assays

• Assess P58IPK localization changes during infection using immunofluorescence

• Quantify P58IPK expression levels in response to viral infection using Western blotting

Studies have demonstrated that P58IPK antibodies can detect the endogenous protein in human, mouse, and monkey samples, making them versatile tools across different experimental models . For Western blotting applications, a dilution of 1:1000 is typically recommended .

What experimental models are suitable for studying P58IPK function?

Several experimental models have proven valuable for studying P58IPK function:

• P58IPK knockout mice: These models have revealed increased lung pathology, immune cell apoptosis, PKR activation, and mortality following influenza virus infection . Transcriptional profile analysis of these mice showed increased expression of genes associated with cell death, immune response, and inflammation .

• P58IPK-/- mouse embryonic fibroblasts (MEFs): These cells demonstrate increased eIF2α phosphorylation and decreased influenza virus mRNA translation compared to wild-type cells . This model has been instrumental in establishing that P58IPK regulates influenza virus mRNA translation through a PKR-mediated mechanism .

• Mathematical modeling: Computational approaches have been developed to examine the P58IPK pathway and investigate the temporal behavior of this biological system. These models accurately predict viral and host protein levels at extended time points and help identify processes that may be targeted to inhibit virus replication .

How can researchers investigate P58IPK interactions with viral proteins?

To investigate P58IPK interactions with viral proteins, researchers can employ several advanced methodologies:

• Protein binding assays: Studies have mapped specific binding domains between P58IPK and viral proteins. For example, P58IPK has been shown to harbor an NP binding site spanning N-terminal TPR subdomains I and II, while the Hsp40 binding site is mapped to the TPR subdomain II . Competitive binding assays can determine relative binding affinities, as demonstrated in studies showing that NP binds P58IPK with higher affinity than Hsp40 .

• Site-directed mutagenesis: Creating NP mutants deficient in binding to either P58IPK or RPS19 has demonstrated that both interactions are necessary for PKR inhibition, highlighting the importance of selective engagement of P58IPK to the 40S ribosomal subunit .

• Ribosomal recruitment assays: These can be used to demonstrate the selective recruitment of the NP-P58IPK complex to the 40S ribosomal subunit, which is crucial for understanding the spatial regulation of PKR inhibition .

What approaches can be used to study the kinetics of the P58IPK pathway during viral infection?

The kinetics of the P58IPK pathway during viral infection can be studied using:

• Time-course experiments: Monitoring phosphorylation levels of PKR and eIF2α at different time points post-infection provides insights into the temporal dynamics of P58IPK activation and its downstream effects .

• Mathematical modeling: Computational models can predict the rapid activation of P58IPK in response to influenza virus infection, which delays and reduces maximal PKR and eIF2α phosphorylation . These models can also simulate long-term viral infection kinetics and correlate various parameters with viral protein synthesis .

• Dose-response studies: Mathematical models suggest that P58IPK activation is dependent on infectious dose, providing a potential experimental avenue for investigating threshold effects in the pathway .

How should researchers design experiments to study P58IPK in different viral infection models?

When designing experiments to study P58IPK in different viral infection models, researchers should consider:

• Virus selection: Different viruses interact with the PKR pathway in distinct ways. While influenza virus and vaccinia virus are particularly sensitive to translational control through eIF2α phosphorylation, reovirus shows different behavior in P58IPK-deficient models . Comparative studies with multiple viruses can provide broader insights into P58IPK function.

• Control selection: Appropriate controls should include:

  • Wild-type vs. P58IPK knockout cells/animals

  • PKR knockout models to distinguish P58IPK effects mediated through PKR versus other pathways

  • PERK knockout models to evaluate the contribution of this alternative P58IPK target

• Endpoint selection: Researchers should measure multiple parameters to fully characterize P58IPK function:

  • eIF2α phosphorylation status

  • Viral mRNA translation efficiency

  • Viral protein synthesis rates

  • Viral titers

  • Host cell viability and apoptosis markers

What are the key considerations when interpreting P58IPK antibody results in infection studies?

When interpreting results from P58IPK antibody experiments in infection studies, researchers should consider:

• Activation status vs. expression level: Post-translational activation of P58IPK may not always correlate with changes in total protein level. Additional assays such as protein-protein interaction studies may be necessary to assess functional activation .

• Temporal dynamics: The rapid activation of P58IPK following infection suggests that early time points are critical for capturing the initial events in the pathway .

• Cell-type specificity: P58IPK's effects may vary between different cell types, particularly between those with robust versus weak interferon responses .

• Potential compensatory mechanisms: In chronic knockout models, compensatory pathways may develop that mask some aspects of P58IPK function. Acute knockdown or inhibition approaches may provide complementary insights .

How can researchers address potential contradictions in P58IPK antibody data?

Researchers may encounter contradictory results when working with P58IPK antibodies. These challenges can be addressed by:

• Antibody validation: Verify antibody specificity using P58IPK knockout controls to ensure signals are specific . Western blotting should show a clear band at 58 kDa that is absent in knockout samples.

• Multiple detection methods: Combine Western blotting with immunofluorescence or flow cytometry to confirm findings from multiple angles.

• Context consideration: P58IPK function changes depending on the cellular context. For example, its role in viral infection may differ from its role in endoplasmic reticulum stress responses. Researchers should carefully define the specific pathway being studied .

• Pathway redundancy analysis: When results appear contradictory, investigate potential redundant mechanisms that might compensate for P58IPK loss. For instance, cells might upregulate alternative PKR inhibitors when P58IPK is absent .

What experimental controls are essential when working with P58IPK antibodies?

Essential controls when working with P58IPK antibodies include:

• Positive control: Samples known to express high levels of P58IPK, such as pancreatic or testicular tissue extracts .

• Negative control: P58IPK knockout or knockdown samples to confirm antibody specificity.

• Loading control: Probing for housekeeping proteins to ensure equal loading, particularly important when comparing expression levels across different conditions.

• Activation controls: When studying P58IPK activation, include controls for both inactive (Hsp40-bound) and active states, such as samples treated with ER stress inducers known to activate P58IPK .

• Species cross-reactivity verification: Verify that the selected antibody recognizes P58IPK in the species being studied, as cross-reactivity may vary between human, mouse, and monkey samples .

How has our understanding of P58IPK function evolved in recent viral pathogenesis research?

Recent developments in P58IPK research have refined our understanding of its function in viral pathogenesis:

• Dual role in host defense: Recent studies have reframed P58IPK as a "cellular inhibitor of the host defense" (CIHD), as it can both promote viral replication and protect the host from excessive inflammation and cell death during infection . This nuanced view suggests that P58IPK may have evolved to balance immediate antiviral defense with longer-term survival of the host.

• Virus-specific effects: While P58IPK deficiency decreases influenza virus and vesicular stomatitis virus replication, it enhances reovirus yields, indicating virus-specific interactions with the P58IPK pathway .

• Mathematical modeling contributions: Computational approaches have provided insights into the temporal dynamics of P58IPK activation and helped identify key regulatory steps that might be targeted to modulate viral replication .

What emerging applications exist for P58IPK antibodies in studying viral host interactions?

Emerging applications for P58IPK antibodies in viral host interaction studies include:

• Single-cell analysis: Combining P58IPK antibodies with single-cell technologies to understand cell-to-cell variability in the PKR response during infection.

• Live-cell imaging: Development of non-disruptive labeling techniques to track P58IPK dynamics in real-time during viral infection.

• Therapeutic target identification: Using P58IPK antibodies to screen for small molecules that could modulate P58IPK function, potentially as antiviral therapeutics.

• Cross-species pathogenesis studies: Utilizing the cross-reactivity of certain P58IPK antibodies across human, mouse, and monkey samples to conduct comparative studies of viral pathogenesis mechanisms across species .