Recombinant Rabbit Calcium-independent phospholipase A2-gamma (PNPLA8)

What is PNPLA8 and how does it differ from other phospholipase enzymes?

PNPLA8 is a member of the patatin-like phospholipase domain-containing protein family. Unlike calcium-dependent phospholipases such as PLA2G4A (cPLA2-α), PNPLA8 belongs to the calcium-independent isoforms (iPLA2) subgroup . It plays a critical role in phospholipid metabolism by catalyzing the hydrolysis of glycerophospholipids, particularly affecting phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and glycerophosphocholine (GPC) .

PNPLA8 contributes to arachidonic acid release and subsequent eicosanoid production, which activates important signaling pathways including PI3K/Akt/GSK3β and MAPK cascades . This differs from the calcium-dependent phospholipase A2-alpha (PLA2G4A), which requires calcium for activation and undergoes phosphorylation and translocation to the plasma membrane during activation .

What are the optimal conditions for detecting PNPLA8 in experimental settings?

For detecting PNPLA8 in experimental settings, researchers should consider:

• Antibody selection: Rabbit recombinant monoclonal antibodies have proven effective for detecting phospholipase enzymes in Western blotting (WB) and immunohistochemistry-paraffin (IHC-P) applications .

• Sample types: Validated antibodies typically work with mouse, rat, and human samples . When working with rabbit PNPLA8, species cross-reactivity should be verified.

• Detection methods:

  • Western blotting: Effective for confirming protein expression or deletion

  • Immunofluorescence: Valuable for visualizing subcellular localization

  • IHC-P: Suitable for tissue section analysis

• Controls: Include positive controls (tissues known to express PNPLA8) and negative controls (PNPLA8 knockout samples) to validate specificity .

What cellular processes are regulated by PNPLA8 activity?

PNPLA8 regulates several critical cellular processes:

How can CRISPR/Cas9 genome editing be effectively applied to study PNPLA8 function?

CRISPR/Cas9 genome editing has proven valuable for studying PNPLA8 function through the following approach:

• sgRNA design and targeting strategy: Design sgRNAs targeting the first two exons of PNPLA8 to ensure complete functional knockout .

• Delivery method: Introduce the designed sgRNA and Cas9 protein into iPSC lines using established transfection protocols .

• Knockout validation:

  • Confirm homozygous truncating variants through sequencing

  • Validate complete deletion of the 77 kDa PNPLA8 protein by immunoblotting

• Experimental applications: Use PNPLA8 knockout iPSCs to generate cerebral organoids for studying neurodevelopmental phenotypes .

This approach has successfully revealed PNPLA8's critical role in brain development, particularly in the formation of basal radial glial cells and upper-layer neurons, demonstrating the power of CRISPR/Cas9 for functional studies of this enzyme .

What are the best approaches for lipidomic analysis in PNPLA8 research?

Lipidomic analysis is essential for understanding PNPLA8's impact on phospholipid metabolism:

• Liquid chromatography/mass spectrometry (LC/MS): The gold standard for comprehensive phospholipid profiling, enabling quantification of multiple phospholipid species simultaneously .

• Key phospholipid targets for analysis:

  • Lyso-phosphatidylcholine (Lyso-PChol)

  • Phosphatidylcholine (PChol)

  • Phosphatidylethanolamine (PE)

  • Phosphatidylserine (PS)

  • Phosphatidylinositol (PI)

• Data analysis and visualization:

  • Calculate fold changes between experimental conditions

  • Plot significant changes in individual phospholipid species

  • Summarize changes per phospholipid class

• Experimental design considerations:

  • Include appropriate controls (wild-type vs. PNPLA8-deficient)

  • Perform experiments in triplicate for statistical validity

  • Consider time-dependent changes in phospholipid profiles

How can researchers effectively silence PNPLA8 expression in experimental models?

Based on successful approaches with related phospholipases, researchers should consider:

• siRNA-mediated gene silencing:

  • Design siRNAs specifically targeting PNPLA8 mRNA sequences

  • Optimize transfection conditions for the specific cell type

  • Validate knockdown efficiency through Western blot and qPCR

• Functional validation:

  • Assess impact on viral protein levels (if studying viral replication)

  • Measure virus production and viral RNA genomes

  • Confirm target protein expression levels

• Rescue experiments:

  • Add exogenous products of PNPLA8 activity to demonstrate specificity

  • Assess restoration of phenotypes to validate mechanisms

• Alternative approaches:

  • CRISPR/Cas9 knockout for complete gene deletion

  • Small molecule inhibitors targeting phospholipase activity (with appropriate specificity controls)

What is the role of PNPLA8 in neurological development and related pathologies?

PNPLA8 plays a critical role in neurological development, with significant implications for brain pathologies:

• Genetic basis of PNPLA8-related disorders:

  • Biallelic null variants in PNPLA8 cause a spectrum of clinical features

  • Complete loss of PNPLA8 is associated with developmental and epileptic-dyskinetic encephalopathy (DEDE) and congenital or progressive microcephaly

• Impact on brain development (based on cerebral organoid studies):

  • PNPLA8 loss reduces basal radial glial cells (bRGCs) in the subventricular zone

  • Significantly fewer upper-layer neurons develop in PNPLA8-deficient organoids

  • No significant effect on deep-layer neurons or ventricular zone size

• Molecular mechanisms:

  • Spatial transcriptomic analysis shows downregulation of bRGC-related gene sets

  • Lipidomic analysis reveals decreased lysophosphatidic acid, lysophosphatidylethanolamine, and phosphatidic acid

  • Disturbed phospholipid metabolism underlies neurodevelopmental abnormalities

How does PNPLA8 contribute to phospholipid reprogramming in cancer?

PNPLA8 has emerged as a key regulator of phospholipid metabolic reprogramming in cancer, particularly in triple-negative breast cancer (TNBC):

• Expression pattern in cancer:

  • PNPLA8 is overexpressed in TNBC cell lines

  • Higher expression observed in breast cancer patient tissues compared to normal tissues

• Functional roles in cancer biology:

  • Regulates cancer cell viability

  • Controls cell migration capacity

  • Contributes to antioxidative responses

  • Silencing PNPLA8 disrupts these cancer-promoting properties

• Phospholipid metabolic effects:

  • PNPLA8 silencing disrupts phospholipid metabolic reprogramming in TNBC

  • Particularly affects levels of PG, PC, LPC, and GPC

  • Promotes arachidonic acid and eicosanoid production

• Signaling pathway connections:

  • Activates PI3K/Akt/Gsk3β signaling

  • Stimulates MAPK pathway activity

  • These pathways support cancer cell proliferation and survival

These findings suggest PNPLA8 could be developed as a novel molecular treatment target for TNBC through disruption of cancer-specific phospholipid metabolism .

What mechanisms might explain PNPLA8's role in viral replication processes?

While the search results don't specifically address PNPLA8 in viral replication, related phospholipase A2 enzymes provide insights into potential mechanisms:

• Phospholipid remodeling during infection:

  • Viral infection elevates phospholipase A2 activity

  • Lyso-PChol shows significant increase (up to 2.9-fold) during West Nile virus infection

  • Other phospholipids (PI, PChol, PE) show altered levels

• Enzyme recruitment and activation:

  • PLA2G4A undergoes translocation from cytoplasm to cell periphery during infection

  • Phosphorylation of PLA2G4A increases during infection

  • Subpopulations of phospholipase enzymes are sequestered to viral protein compartments

• Functional relevance:

  • Inhibition of PLA2 activity reduces viral replication

  • Addition of exogenous lyso-PChol rescues virus production

  • Phospholipase products are required for viral protein synthesis

• Potential PNPLA8-specific mechanisms:

  • PNPLA8 may alter membrane compositions required for viral replication complexes

  • Its calcium-independent activity could provide constitutive membrane remodeling

  • Connection to arachidonic acid cascade may link to inflammatory responses during infection

What are the applications of cerebral organoids in studying PNPLA8-related neurological disorders?

Cerebral organoids have emerged as powerful models for studying PNPLA8's role in brain development:

• Advantages of the cerebral organoid system:

  • Recapitulates human-specific aspects of brain development

  • Allows for longitudinal studies of developmental processes

  • Enables manipulation of specific genes in human neural context

• Experimental approaches with PNPLA8-deficient organoids:

  • Generation from CRISPR/Cas9-edited iPSCs with PNPLA8 knockout

  • Cultivation for various time periods (4, 8, and 12 weeks)

  • Immunostaining for neural progenitor and neuronal markers

• Key findings from organoid models:

  • SVZ area is reduced in PNPLA8 KO organoids at 12 weeks

  • SATB2+ upper-layer neurons are significantly decreased

  • CTIP2+ deep-layer neurons remain unaffected

  • These findings correspond to clinical microcephaly phenotypes

• Advanced analytical techniques:

  • Spatial transcriptomic analysis targeting apical radial glial cells

  • Lipidomic analysis to characterize disrupted phospholipid metabolism

  • Immunostaining for markers of neural progenitors (SOX2+, TBR2+) and neurons (SATB2+, CTIP2+)

How might targeting PNPLA8 lead to novel therapeutic approaches for cancer?

PNPLA8 shows promise as a therapeutic target based on several lines of evidence:

• Overexpression pattern:

  • PNPLA8 is overexpressed in TNBC cell lines and tissues

  • This expression pattern provides a potential cancer-specific target

• Critical roles in cancer cell biology:

  • Regulates cancer cell viability

  • Controls migration capacity

  • Contributes to antioxidative responses

  • These functions are essential for cancer progression

• Signaling pathway connections:

  • PNPLA8 promotes arachidonic acid and eicosanoid production

  • These lipid mediators activate PI3K/Akt/Gsk3β and MAPK signaling

  • Targeting PNPLA8 could disrupt these oncogenic pathways

• Potential therapeutic strategies:

  • Small molecule inhibitors targeting PNPLA8 enzymatic activity

  • siRNA or antisense oligonucleotides for gene silencing

  • Blocking downstream lipid mediator signaling

  • Combination with existing therapies targeting PI3K/Akt or MAPK pathways

Comprehensive lipid profiling revealed that targeting PNPLA8 disrupts phospholipid metabolic reprogramming in TNBC, suggesting it could be developed as a novel molecular treatment target with potential applications in diagnostics and therapeutics .

What are the key considerations when selecting antibodies for PNPLA8 detection?

When selecting antibodies for PNPLA8 detection, researchers should consider:

• Antibody type and source:

  • Rabbit recombinant monoclonal antibodies offer high specificity

  • Validated antibodies like those for related phospholipases have proven effective in Western blotting (WB) and immunohistochemistry-paraffin (IHC-P)

• Applications and validation:

  • Verify antibody suitability for specific applications (WB, IHC-P, IF)

  • Check validation for species of interest (mouse, rat, human)

  • Review publication citations to confirm reliability

• Target specificity:

  • Ensure antibodies specifically recognize PNPLA8 (85/88 kDa) and not related proteins

  • Check for cross-reactivity with other PNPLA family members

  • Consider epitope locations relative to functional domains

• Alternative names awareness:

  • PNPLA8 may be listed under alternative nomenclature:

    • iPLA2-gamma

    • IPLA2-gamma

    • Calcium-independent phospholipase A2 gamma

    • Group VIB phospholipase A2

What are the methodological challenges in measuring PNPLA8 enzymatic activity?

Measuring PNPLA8 enzymatic activity presents several methodological challenges:

• Distinguishing from other phospholipases:

  • Multiple PLA2 isoforms may be present in cellular samples

  • Calcium-independent activity helps distinguish from cPLA2 enzymes

  • Specific inhibitors may be needed to isolate PNPLA8 activity

• Activity assay considerations:

  • Use fluorogenic or radiometric substrates for direct enzymatic activity

  • Measure release of free fatty acids or lysophospholipids

  • Include appropriate positive and negative controls

• Lipidomic analysis approach:

  • LC/MS analysis provides comprehensive phospholipid profiling

  • Focus on known PNPLA8 substrates and products

  • Quantify fold changes in phospholipid species before and after manipulation

• Cellular localization impacts:

  • PNPLA8 activity may be compartmentalized within cells

  • Subcellular fractionation may be necessary for accurate activity measurement

  • Translocation during activation can affect measurement results