Recombinant Mouse Phosducin-like protein 3 (Pdcl3)

What is the molecular structure and evolutionary significance of Pdcl3?

Pdcl3 belongs to the photoreceptor family characterized by a thioredoxin-like structural domain with significant evolutionary conservation across species. The protein forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. This interaction is critical for understanding its cellular functions, as Pdcl3 participates in protein folding and quality control mechanisms within cells . The thioredoxin-like domain enables specific protein-protein interactions that are essential for its regulatory roles in various cellular processes, including tubulin folding.

What are the primary biological functions of Pdcl3 in normal cellular physiology?

Pdcl3 plays crucial roles in angiogenesis and apoptosis regulation in normal cellular physiology. Its most well-characterized function involves regulating the balance between α and β tubulin subunits, which is essential for proper microtubule assembly and cytoskeletal dynamics . Experimental evidence indicates that optimal Pdcl3 levels are necessary for maintaining cellular homeostasis, as either overexpression or silencing disrupts normal cellular functions. Research demonstrates that Pdcl3 interacts with the CCT chaperonin complex to regulate protein folding, particularly for cytoskeletal proteins, suggesting its importance in maintaining cellular architecture and division processes.

What methods are recommended for analyzing Pdcl3 expression patterns in experimental models?

For comprehensive analysis of Pdcl3 expression, researchers should employ multiple complementary techniques:

• RNA-seq analysis provides transcriptome-wide context for Pdcl3 expression across different tissues or experimental conditions

• Quantitative RT-PCR offers precise quantification of Pdcl3 mRNA levels

• Immunohistochemistry (IHC) and immunofluorescence are essential for validating protein expression and localization in tissue samples

• Western blotting quantifies total protein levels and can detect potential post-translational modifications

For optimal results, validation across multiple techniques is recommended. In glioma research, for example, both mRNA expression through qRT-PCR and protein expression through IHC have been employed to confirm differential expression patterns across tumor grades . The combination of these approaches provides more robust evidence than any single method alone.

What are effective strategies for manipulating Pdcl3 expression in functional studies?

Researchers have successfully employed several approaches to modulate Pdcl3 expression:

• Overexpression systems using plasmid vectors containing the Pdcl3 gene have demonstrated that increased Pdcl3 levels promote an imbalance of α and β tubulin subunits, leading to microtubule disassembly and cell death

• RNA interference through siRNA targeting Pdcl3 results in increased RhoA-dependent actin filament formation, focal adhesion assembly, and dramatic morphological changes toward an elongated fibroblast-like phenotype

• Rescue experiments combining knockdown with transient overexpression can confirm specificity of observed phenotypes, though careful titration is necessary as complete rescue remains challenging due to dosage sensitivity

When designing such experiments, researchers should consider dose-dependent effects, as both overexpression and silencing produce distinct cellular phenotypes, suggesting Pdcl3 levels are finely balanced in normal cells.

How does Pdcl3 expression vary across different cancer types and what methodologies best detect these differences?

Pdcl3 exhibits significant expression variation across cancer types, with consistent upregulation in multiple cancers. Comprehensive analysis using TIMER, CPTAC, and TCGA databases revealed:

• Significant upregulation in 22 cancer types, including BRCA, CHOL, COAD, ESCA, HNSC, LIHC, LUAD, LUSC, PRAD, STAD, and UCEC

• Marked reduction in KICH and PCPG

• Correlation with higher tumor grade in multiple cancers, particularly in glioma and liver hepatocellular carcinoma

For reliable detection of these differences, researchers should combine database mining with experimental validation. In glioma research, ROC curve analysis demonstrated excellent discriminatory power with AUC values ranging from 0.692 to 0.905 across different cohorts, confirming Pdcl3's potential as a diagnostic biomarker .

What is the prognostic value of Pdcl3 expression in cancer and how can it be assessed?

High Pdcl3 expression correlates with poorer clinical outcomes across multiple cancer types:

For clinical assessment, several approaches have demonstrated efficacy:

• Kaplan-Meier survival analysis comparing high vs. low expression groups

• ROC curve analysis for diagnostic accuracy (AUC values)

• Multivariate Cox regression to establish independent prognostic value

• Nomogram development for individualized prediction

A nomogram incorporating Pdcl3 expression with other clinical factors achieved a C-index of 0.864 for glioma prognosis prediction, with excellent calibration for 1-, 3-, and 5-year OS prediction (respective AUC values of 0.905, 0.919, and 0.912) .

What techniques are recommended for assessing Pdcl3's interaction with tubulin and the cytoskeleton?

To effectively characterize Pdcl3-tubulin interactions, researchers should employ a multi-faceted approach:

• Co-immunoprecipitation assays to confirm physical interaction between Pdcl3, CCT chaperonin, and tubulin in cellular contexts

• Fluorescence microscopy with co-staining for Pdcl3 and tubulin to assess co-localization patterns

• In vitro microtubule assembly/disassembly assays following Pdcl3 modulation to directly measure functional impact

• Live-cell imaging with fluorescently tagged tubulin to monitor real-time effects of Pdcl3 manipulation on microtubule dynamics

Research has demonstrated that overexpression of Pdcl3 promotes an imbalance of α and β tubulin subunits, leading to microtubule disassembly and cell death . This suggests that quantitative assessment of tubulin subunit ratios could serve as a useful readout for Pdcl3 function.

How can researchers accurately quantify morphological changes resulting from Pdcl3 modulation?

Accurate quantification of morphological changes requires systematic approaches:

• Automated image analysis software (e.g., CellProfiler, ImageJ) to measure:

  • Cell elongation ratio (length/width)

  • Cell area and perimeter

  • Fractal dimension analysis for complexity assessment

  • Focal adhesion number, size, and distribution

• Quantitative immunofluorescence for:

  • Actin filament organization (stress fiber formation)

  • Focal adhesion markers (vinculin, paxillin)

  • RhoA activation status

  • Phosphorylated MAPK levels

When analyzing Pdcl3 knockdown effects, researchers observed dramatic elongated fibroblast-like morphological changes, along with increased RhoA-dependent actin filament formation and focal adhesion assembly . These changes were accompanied by increased phosphorylated MAPK, suggesting a signaling mechanism connecting Pdcl3 to cytoskeletal remodeling.

What mechanisms explain Pdcl3's regulatory effects on microtubule dynamics?

Pdcl3 exerts its effects on microtubule dynamics through several interconnected mechanisms:

• Regulation of tubulin folding by forming a ternary complex with the CCT chaperonin and tubulin

• Maintenance of proper α/β tubulin subunit balance, which is critical for microtubule polymerization

• Potential regulation of tubulin post-translational modifications that affect microtubule stability

Experimental evidence demonstrates that overexpression of Pdcl3 promotes an imbalance of α and β tubulin subunits, leading to microtubule disassembly and ultimately cell death . This indicates that precise control of Pdcl3 levels is essential for proper microtubule function, with both excessive and insufficient levels causing distinct cytoskeletal defects.

How does Pdcl3 modulation affect actin cytoskeleton organization and what signaling pathways are involved?

Pdcl3 modulation has significant impacts on actin cytoskeleton organization through several signaling pathways:

• RNA silencing of Pdcl3 increases RhoA-dependent actin filament formation and focal adhesion assembly

• Pdcl3 knockdown promotes MAPK phosphorylation, which is associated with focal adhesion maturation

• The elongated fibroblast-like phenotype observed after Pdcl3 silencing suggests effects on cell polarity pathways

These observations indicate that while Pdcl3's direct interaction with tubulin is well-established, it also influences actin cytoskeleton organization, potentially through indirect mechanisms involving RhoA and MAPK signaling cascades. This suggests Pdcl3 may function as an integrator of microtubule and actin cytoskeleton regulation, warranting further investigation into cross-talk mechanisms.

What is the relationship between Pdcl3 expression and immune cell infiltration in cancer microenvironments?

Analysis of Pdcl3's relationship with immune infiltration has revealed significant associations:

• TIMER 2.0 database analysis shows a strong negative correlation between Pdcl3 expression and macrophage infiltration (Rho = -0.481, p = 2.13e-21) in liver cancer

• High Pdcl3 expression groups show significantly lower macrophage infiltration compared to low expression groups

• In cases with high Pdcl3 expression, decreased macrophage infiltration correlates with adverse prognosis, while this correlation is not observed in low Pdcl3 expression cases

These findings suggest that Pdcl3 may influence tumor progression partly through modulating the immune microenvironment, particularly by affecting macrophage recruitment or survival. For researchers investigating cancer immunobiology, Pdcl3 represents a potential link between cytoskeletal regulation and immune cell behavior.

Which immune-related biological processes are associated with Pdcl3 function?

Gene ontology and pathway enrichment analyses have identified several immune-related processes associated with Pdcl3:

• Biological process (BP) analysis shows enrichment in:

  • Humoral immune response

  • Immunoglobulin-mediated immune response

  • B-cell-mediated immunity

• Cellular component (CC) analysis reveals associations with:

  • Immunoglobulin complexes

  • Circulating immunoglobulin complexes

• Molecular function (MF) analysis indicates involvement in:

  • Immunoglobulin receptor binding

These associations suggest that beyond its role in cytoskeletal regulation, Pdcl3 may have broader functions in immune system modulation, particularly in B-cell-mediated immunity and antibody production pathways. This presents an intriguing area for further investigation, especially in contexts where cytoskeletal dynamics intersect with immune cell function.