PFDN6 Human

What is PFDN6 and what is its primary function in human cells?

PFDN6, also known as H2-KE2, HKE2, KE-2, or PFD6, is a subunit of the heteromeric prefoldin complex that functions as a molecular chaperone in human cells . Its primary role is to assist in the proper folding of newly synthesized proteins, particularly cytoskeletal components. PFDN6 specifically chaperones nascent actin and alpha- and beta-tubulin chains pending their transfer to the cytosolic chaperonin containing TCP1 (CCT) complex . This function is critical because it prevents protein misfolding and aggregation during protein synthesis, ensuring proper protein maturation in an environment where there are many competing pathways for nonnative proteins.

The molecular mechanism involves PFDN6 binding specifically to cytosolic chaperonin (c-CPN) to facilitate the transfer of target proteins, thus promoting proper protein folding and preventing potentially detrimental aggregation . This chaperoning activity is essential for maintaining cellular proteostasis and supporting cytoskeletal dynamics.

What methodologies are used to study PFDN6 expression in tissues?

Researchers employ several complementary techniques to analyze PFDN6 expression:

• Immunohistochemistry (IHC): This technique allows visualization of PFDN6 protein in tissue sections, enabling assessment of expression levels and localization patterns in normal versus diseased tissues (e.g., cancer) .

• Western Blotting: A quantitative method to determine PFDN6 protein levels using specific antibodies, often with GAPDH as an internal control . This technique is particularly useful for comparing expression levels between different cell lines or tissue samples.

• Real-time PCR (qRT-PCR): This allows quantification of PFDN6 mRNA expression levels, providing insights into transcriptional regulation .

• Transcriptome Sequencing: Used to analyze gene expression profiles, including PFDN6, and to identify differentially expressed genes after PFDN6 knockdown or overexpression .

• Bioinformatics Analysis: Various databases such as TCGA, Oncomine, and analytical tools are used to examine PFDN6 expression across different cancer types and correlate expression with clinical parameters .

For optimal results, researchers should implement multiple methods to validate PFDN6 expression findings, as each technique provides different but complementary information about gene and protein expression.

What is the role of PFDN6 in colorectal cancer progression?

PFDN6 has been identified as a potential oncogenic factor in colorectal cancer (CRC). Research has demonstrated that PFDN6 expression is significantly elevated in CRC tissues compared to para-carcinoma tissues, with approximately 51.9% of tumor samples showing high expression compared to only 1.0% in para-carcinoma tissues (p<0.001) . This striking differential expression suggests a role in carcinogenesis.

Functional studies have revealed that PFDN6 contributes to several hallmarks of cancer:

• Cell Proliferation: PFDN6 knockdown reduces tumor cell numbers in CRC cell lines (HCT-116 and RKO) .

• Apoptosis Regulation: Silencing PFDN6 promotes apoptosis in CRC cells, indicating its role in cell survival mechanisms .

• Migration and Invasion: PFDN6 knockdown significantly inhibits the migration and invasion capabilities of CRC cells, suggesting its involvement in metastatic processes .

Mechanistically, PFDN6 appears to regulate CRC development by targeting ZNF575, as revealed by transcriptome sequencing analysis of differentially expressed genes following PFDN6 knockdown . These findings collectively position PFDN6 as a potential therapeutic target for CRC treatment.

How can researchers experimentally manipulate PFDN6 expression in cell models?

Researchers can modulate PFDN6 expression through several established methods:

• RNA Interference (RNAi): Short hairpin RNA (shRNA) targeting PFDN6 can be designed and delivered via viral vectors (e.g., BR-V-108 vector) to achieve stable knockdown. Multiple shRNA constructs (e.g., shPFDN6-1, shPFDN6-2, shPFDN6-3) should be tested to identify the most efficient one . For example, in CRC research, shPFDN6-3 achieved optimal knockdown efficiency (p<0.01) .

• Plasmid-based Overexpression: PFDN6 overexpression can be achieved by cloning the PFDN6 coding sequence into expression vectors. These constructs can be designed with epitope tags for easier detection .

• CRISPR-Cas9 Gene Editing: For complete knockout or precise genomic modifications of the PFDN6 gene.

The efficiency of manipulation should be verified through:

• qRT-PCR for mRNA level changes

• Western blot for protein level alterations

• Immunofluorescence (IF) to visualize changes in cellular localization and expression

These genetic manipulation approaches enable researchers to investigate the functional consequences of PFDN6 alteration in cellular processes related to cancer progression.

What functional assays are appropriate for evaluating PFDN6's role in cancer research?

To comprehensively assess PFDN6's impact on cancer-related phenotypes, researchers should employ multiple functional assays:

• Cell Proliferation Assays:

  • Celigo image cytometry for generating cell proliferation curves

  • Cell counting at different time points after PFDN6 knockdown or overexpression

• Apoptosis Detection:

  • Flow cytometry with Annexin V/PI staining

  • TUNEL assay for detecting DNA fragmentation

• Cell Migration Assays:

  • Wound healing assay: Cells are seeded at 5×10⁴ cells/well in 96-well plates, and migration into the wound area is monitored after creating a scratch

  • Transwell migration assay

• Cell Invasion Assays:

  • Matrigel-coated transwell chambers to assess invasive capacity

• In vivo Xenograft Models:

  • Subcutaneous injection of PFDN6-manipulated cancer cells into immunodeficient mice to evaluate tumor growth

  • Metastasis models to assess the impact on cancer spread

• Molecular Mechanism Exploration:

  • Transcriptome sequencing to identify differentially expressed genes (DEGs) after PFDN6 manipulation

  • Analysis of DEGs using criteria such as |logFC|>log2(1.5) and p<0.05 (using Benjamini-Hochberg correction)

  • Pathway analysis to identify affected signaling networks

These assays collectively provide a comprehensive understanding of PFDN6's functional role in cancer biology.

How can recombinant PFDN6 be produced and purified for research purposes?

Production of high-quality recombinant PFDN6 for research applications involves several steps:

• Expression System Selection: E. coli is commonly used for PFDN6 expression due to its simplicity and high yield .

• Vector Design:

  • PFDN6 cDNA is cloned into an expression vector with a His-tag at the N-terminus to facilitate purification

  • The construct typically includes appropriate promoters (e.g., T7) and selection markers

• Expression Conditions:

  • Optimization of induction parameters (temperature, IPTG concentration, duration)

  • Growth in suitable media for protein production

• Purification Protocol:

  • Lysis of bacteria in appropriate buffer

  • Purification using conventional chromatography techniques:

    • Immobilized metal affinity chromatography (IMAC) utilizing the His-tag

    • Further purification steps may include ion exchange or size exclusion chromatography

  • Final product should achieve >95% purity as verified by SDS-PAGE

• Characterization:

  • Molecular weight confirmation using MALDI-TOF (expected ~30.9 kDa for His-tagged product)

  • Functional assays to verify biological activity

  • Protein concentration determination (e.g., Bradford assay)

• Storage Recommendations:

  • Short-term storage at +4°C (1-2 weeks)

  • Long-term storage at -20°C or -70°C

  • Aliquoting to avoid repeated freeze-thaw cycles

  • Recommended storage buffer: 20 mM Tris-HCl buffer (pH 8.0) containing 0.1M NaCl, 1mM DTT, 10% glycerol

This methodology yields pure, functional PFDN6 protein suitable for various research applications including structural studies, protein-protein interaction analyses, and functional assays.

What bioinformatics approaches are valuable for studying PFDN6 in cancer research?

Several bioinformatics strategies have proven effective for investigating PFDN6 in cancer contexts:

These bioinformatics tools collectively provide a comprehensive framework for investigating PFDN6's role in cancer initiation, progression, and potential as a therapeutic target or biomarker.

How is PFDN6 expression correlated with clinical features in cancer patients?

Research has demonstrated that PFDN6 expression correlates with several clinical parameters in cancer patients:

• Expression Pattern: Immunohistochemistry analysis of colorectal cancer tissues has shown significantly higher PFDN6 expression in tumor tissues compared to para-carcinoma tissues. Specifically, 51.9% of tumor tissues showed high PFDN6 expression versus only 1.0% in para-carcinoma tissues (p=0.001) .

• Correlation Table: The following summarizes PFDN6 expression patterns in colorectal cancer:

This stark difference suggests PFDN6 upregulation is highly specific to the cancerous state .

• Stage Correlation: While the specific data for PFDN6 is limited in the search results, studies of related prefoldin family members (PFDN1-4) have shown correlation with advanced clinicopathologic features in hepatocellular carcinoma .

These clinical correlations suggest that PFDN6 expression analysis could have potential utility as a biomarker for cancer diagnosis and prognosis assessment.

What molecular mechanisms underlie PFDN6's role in tumorigenesis?

The molecular mechanisms through which PFDN6 contributes to tumorigenesis are multi-faceted:

Understanding these molecular mechanisms provides potential targets for therapeutic intervention and opportunities for biomarker development in cancer diagnostics and prognostics.

How does PFDN6 compare functionally to other prefoldin family members?

The prefoldin family consists of six subunits (PFDN1-6) that form a hexameric complex, but they exhibit different expression patterns and functional roles in cancer:

This comparative analysis suggests that while the prefoldin family shares core chaperone functions, individual members like PFDN6 may have evolved specialized roles in different cellular contexts and cancer types.

What are best practices for PFDN6 knockdown studies in cancer research?

Effective PFDN6 knockdown studies require careful experimental design and validation:

• shRNA Design and Selection:

  • Design multiple shRNA constructs targeting different regions of PFDN6 mRNA (e.g., shPFDN6-1, shPFDN6-2, shPFDN6-3)

  • Incorporate shRNA sequences into appropriate vectors (e.g., BR-V-108) using restriction sites at both ends

  • Transform into competent cells (e.g., TOP10 E. coli) for plasmid production

  • Test all constructs to identify the one with optimal knockdown efficiency (e.g., in CRC research, shPFDN6-3 achieved best results)

• Delivery Method Selection:

  • Lentiviral transduction for stable knockdown

  • Lipofection for transient knockdown studies

  • Selection of appropriate controls (scrambled shRNA sequences)

• Validation of Knockdown Efficiency:

  • qRT-PCR to verify reduction in PFDN6 mRNA levels

  • Western blot analysis to confirm decreased PFDN6 protein expression (using GAPDH as internal control)

  • Immunofluorescence to visualize reduced PFDN6 protein levels in cells

• Functional Assessment Protocol:

  • Cell counting: Seed cells at appropriate density (e.g., 2000 cells/well in 96-well plates) and monitor growth

  • Migration assays: Use wound healing assay with 5×10⁴ cells/well in 96-well plates

  • Celigo image cytometry for generating accurate cell proliferation curves

• Molecular Mechanism Exploration:

  • Transcriptome analysis to identify genes affected by PFDN6 knockdown

  • Apply stringent criteria for identifying differentially expressed genes (|logFC|>log2(1.5), p<0.05)

These methodological approaches ensure robust and reproducible results when investigating PFDN6's role in cancer biology.

How can researchers effectively analyze PFDN6-dependent gene expression changes?

Comprehensive analysis of PFDN6-dependent gene expression requires a multi-step approach:

• Experimental Setup:

  • Establish stable PFDN6 knockdown or overexpression cell lines alongside appropriate controls

  • Confirm PFDN6 manipulation efficiency at both mRNA and protein levels

  • Use multiple biological replicates to ensure statistical robustness

• RNA Extraction and Quality Control:

  • Extract total RNA using validated methods (e.g., RNeasy kit)

  • Assess RNA quality and integrity using spectrophotometry (e.g., Nanodrop 2000) and gel electrophoresis

  • Ensure RNA samples meet quality thresholds for downstream applications

• Transcriptome Analysis Options:

  • Microarray Analysis: Use platforms such as PrimeView human gene expression array

  • RNA-Seq: For more comprehensive and unbiased transcriptome profiling

  • qRT-PCR: For targeted validation of specific gene expression changes

• Bioinformatics Analysis Pipeline:

  • Identify differentially expressed genes (DEGs) using established statistical methods

  • Apply appropriate thresholds: |logFC|>log2(1.5) and p<0.05 after Benjamini-Hochberg correction

  • Select genes with highest fold change and lowest p-value as potential direct targets

• Functional Annotation:

  • Perform Gene Ontology (GO) analysis to identify enriched biological processes

  • Conduct KEGG pathway analysis to map DEGs to known signaling pathways

  • Use Gene Set Enrichment Analysis (GSEA) with criteria of p<0.05 and FDR q-value<0.25

• Validation of Key Targets:

  • Confirm expression changes of selected target genes (e.g., ZNF575 in colorectal cancer) using qRT-PCR and western blotting

  • Perform functional studies on identified targets to confirm their role in PFDN6-mediated effects

This systematic approach provides a comprehensive understanding of PFDN6-dependent gene expression changes and identifies potential therapeutic targets or biomarkers.

What are promising therapeutic strategies targeting PFDN6 in cancer?

Several potential therapeutic approaches targeting PFDN6 are worth exploring:

• RNA Interference-Based Therapeutics:

  • Development of siRNA or shRNA delivery systems specifically targeting PFDN6

  • Design of lipid nanoparticles or viral vectors to efficiently deliver RNAi molecules to tumor tissues

  • Optimization of RNAi sequences based on knockdown studies that have shown efficacy (e.g., shPFDN6-3)

• Small Molecule Inhibitors:

  • Structure-based drug design targeting PFDN6's interaction with client proteins or other prefoldin subunits

  • High-throughput screening to identify compounds that disrupt PFDN6 function

  • Development of allosteric modulators that alter PFDN6's chaperoning activity

• Combination Therapies:

  • Pairing PFDN6 inhibition with conventional chemotherapeutics to enhance efficacy

  • Targeting PFDN6 alongside its downstream effectors (e.g., ZNF575)

  • Combining PFDN6 inhibition with immunotherapy approaches

• Antibody-Based Approaches:

  • Development of antibody-drug conjugates targeting PFDN6-overexpressing cancer cells

  • Bispecific antibodies linking PFDN6-expressing cells to immune effectors

• Gene Editing Strategies:

  • CRISPR-Cas9 approaches to knock out PFDN6 in localized tumors

  • Development of cancer-specific promoter-driven gene editing systems

These therapeutic strategies require careful evaluation of efficacy, specificity, and potential off-target effects, given PFDN6's normal cellular functions in protein folding. Preliminary studies in colorectal cancer models have demonstrated that PFDN6 knockdown can reduce tumor cell number, promote apoptosis, and inhibit migration and invasion , suggesting therapeutic potential for PFDN6-targeting approaches.

What are the limitations of current PFDN6 research and key knowledge gaps?

Despite progress in understanding PFDN6, several limitations and knowledge gaps remain:

• Structural Understanding:

  • Limited structural characterization of PFDN6 alone and within the prefoldin complex

  • Incomplete understanding of how PFDN6 recognizes and binds client proteins

  • Need for crystallographic or cryo-EM studies of PFDN6 in different functional states

• Cancer Type Specificity:

  • Current research focuses primarily on colorectal cancer , with limited data on PFDN6's role in other cancer types

  • Unclear whether PFDN6's effects are universal across cancers or context-dependent

  • Need for comprehensive pan-cancer analysis of PFDN6 expression and function

• Mechanistic Details:

  • Incomplete understanding of how PFDN6 regulates ZNF575 and other potential targets

  • Limited knowledge of PFDN6's role in specific signaling pathways

  • Unknown post-translational modifications that might regulate PFDN6 function

• Physiological Role:

  • Limited understanding of PFDN6's normal physiological functions beyond protein folding

  • Unclear consequences of long-term PFDN6 inhibition on normal tissues

  • Potential role in other diseases besides cancer (e.g., neurodegenerative disorders)

• Therapeutic Development Challenges:

  • Difficulty in specifically targeting PFDN6 without affecting other prefoldin subunits

  • Unknown potential for resistance mechanisms to PFDN6-targeted therapies

  • Need for biomarkers to identify patients most likely to respond to PFDN6-targeted interventions

Addressing these knowledge gaps will require multidisciplinary approaches combining structural biology, cancer biology, proteomics, and clinical research to fully understand PFDN6's potential as a therapeutic target and biomarker.