Recombinant Rat 6-phosphofructokinase type C (Pfkp)

What is Rat 6-phosphofructokinase type C (Pfkp) and what is its role in cellular metabolism?

Rat 6-phosphofructokinase type C (Pfkp) is a key rate-limiting enzyme in the glycolytic pathway that plays a critical role in cellular energy metabolism. It catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, a crucial step in glycolysis. Pfkp functions as a tetrameric protein and is also known by several other names including PFKF, PFK-C, Phosphohexokinase, and Phosphofructo-1-kinase isozyme C . Dysregulation of Pfkp has been implicated in various diseases, including cancer and metabolic disorders, making it an important target for metabolic research .

What are the structural characteristics of Rat Pfkp?

While the exact structural details of rat Pfkp are not fully described in the search results, information from the human platelet isoform (PFKP) indicates that it exists as a tetramer, as confirmed through transmission electron microscopy . The protein can be divided into catalytic and regulatory domains . The human PFKP structure has been determined in complex with ATP-Mg²⁺ and ADP at 3.1 and 3.4 Å resolution, providing insights that may be relevant to the rat ortholog due to evolutionary conservation . Specific residues like Asn426, Asp564, and Arg319 form important interactions at the catalytic interface in human PFKP, and these structural features likely have parallels in the rat enzyme .

How is Pfkp expression regulated in different cell types?

Pfkp expression appears to be regulated through multiple mechanisms, including transcriptional control. In mouse embryonic stem cells (mESCs), Pfkp is transcriptionally repressed by the transcription factor Stat3, as evidenced by increased Pfkp mRNA, pre-mRNA, and protein levels following Stat3 silencing . During differentiation processes such as embryoid body formation or after leukemia inhibitory factor (LIF) withdrawal, Pfkp expression is upregulated . Additionally, in cancer contexts, ectopic expression of PFKP may depend on DNA methylation and several transcription factors, including members of the KLF and Sp families .

What non-glycolytic functions does Pfkp perform in cellular systems?

Beyond its canonical role in glycolysis, Pfkp demonstrates important non-glycolytic functions, particularly in embryonic stem cell differentiation. Research indicates that Pfkp catalyzes the phosphorylation of Lin41 (also known as TRIM71), which protects Lin41 from proteasomal destruction . This interaction appears to be critical for regulating pluripotency and differentiation. Specifically, while knockdown of other glycolytic enzymes (Hk2, Pfkl) reduced pluripotency markers in mESCs, Pfkp knockdown surprisingly enhanced pluripotency marker expression, especially Sox2 . Pfkp overexpression drove mESCs toward differentiation, downregulating core pluripotency markers and causing morphological changes consistent with differentiation .

How does Pfkp contribute to lineage specification during differentiation?

Pfkp plays a distinctive role in lineage specification during embryonic stem cell differentiation. Transcriptomic analysis comparing control and Pfkp-knockdown differentiated ESC cultures revealed that Pfkp inhibits ectodermal differentiation while promoting endodermal differentiation . When Pfkp was silenced, ectodermal specification genes showed elevated expression while endodermal and mesodermal specification genes were diminished . In practical terms, mESC cultures directed toward endodermal differentiation produced smaller populations of Sox17 and Foxa2-positive cells when Pfkp was silenced, whereas Pfkp-knockdown mESCs generated increased numbers of β-III Tubulin and neurofilament-L-positive cells using a neuronal differentiation protocol .

What is the significance of Pfkp in disease models and potential therapeutic contexts?

Emerging research suggests Pfkp has significant implications in disease contexts. In cancer research, high expression of PFKP correlates with poor prognosis across multiple cancer types, suggesting its potential as a prognostic biomarker . PFKP expression also shows positive correlation with programmed cell death-ligand 1 (PD-L1), indicating potential connections to immune regulation and immunotherapy response .

In osteoarthritis (OA) research, the FUNDC1/PFKP-mediated mitophagy pathway appears important. KD025 treatment induces interaction between PFKP and FUNDC1, enhancing mitophagy and ameliorating OA conditions . This treatment decreased expression of matrix metalloproteinases (MMP3, MMP13) while upregulating aggrecan (ACAN) expression in primary human chondrocytes, suggesting potential therapeutic applications .

What expression systems are optimal for producing recombinant Rat Pfkp?

For high-quality recombinant Rat Pfkp production, baculovirus expression systems have proven effective. According to the search results, the following protocol has been successfully employed:

• Clone Pfkp cDNA into an appropriate vector (e.g., pFastBac HTa)

• Generate baculovirus using expression systems like Bac-to-Bac Expression system

• Infect insect cells (sf21 or Hi5) at a multiplicity of infection of 1 for 48 hours

• Harvest and process cells using appropriate lysis buffer (e.g., 20 mM Tris-HCl pH 7.5, 50 mM potassium phosphate, 1 mM 2-mercaptoethanol, 10% glycerol, 10 mM imidazole, and protease inhibitors)

• Homogenize cells and remove debris by centrifugation

This approach yields tetrameric recombinant Pfkp with activity and regulatory properties similar to the native enzyme .

What are the recommended methods for quantifying Pfkp levels in different biological samples?

For quantitative detection of Pfkp in biological samples, sandwich ELISA is the recommended approach. Commercially available Rat 6-phosphofructokinase type C ELISA kits offer:

• Detection range: 0.312-20 ng/mL

• Sensitivity: approximately 0.177-0.188 ng/mL

• Sample compatibility: serum, plasma, tissue homogenates, cell culture supernatants, and other biological fluids

• Specificity: natural and recombinant rat 6-phosphofructokinase type C

For protein interaction studies, immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) techniques have been successfully employed. A typical protocol includes:

• Use of specific antibodies (e.g., anti-FUNDC1 at 1:200 dilution)

• Overnight incubation at 4°C

• Incubation with protein A/G magnetic beads

• Washing with lysis buffer

• Analysis by Western blotting or mass spectrometry

How can the enzymatic activity of Pfkp be accurately measured in experimental settings?

While the search results don't provide complete details for Pfkp activity assays, evidence from recombinant PFKP studies indicates that enzymatic activity can be measured in cell lysates . The recombinant enzyme shows high cooperativity for fructose-6-phosphate (F6P), high affinity for ATP-Mg²⁺, and high sensitivity to ATP inhibition .

When expressing PFKP tagged with GFP in cell lines (e.g., MTLn3 rat mammary adenocarcinoma cells), PFK1 activity can be measured in cell lysates, and metabolic consequences can be assessed by measuring parameters such as lactic acid excretion . This approach allows for comparing the activity of wild-type versus mutant forms of the enzyme, providing insights into structure-function relationships.

What strategies can address inconsistent Pfkp expression levels in recombinant systems?

When encountering inconsistent expression of recombinant Pfkp, consider optimizing these parameters:

• Expression vector selection: The search results indicate successful use of pFastBac HTa vector with baculovirus expression systems

• Cell type selection: Both sf21 and Hi5 insect cells have been successfully used, with expression at a multiplicity of infection of 1 for 48 hours

• Temperature and time conditions: These should be systematically optimized for your specific experimental setup

• Lysis buffer composition: Use appropriate buffers containing protease inhibitors to protect the expressed protein (e.g., 20 mM Tris-HCl pH 7.5, 50 mM potassium phosphate, 1 mM 2-mercaptoethanol, 10% glycerol, 10 mM imidazole, with complete Protease Inhibitor Cocktail)

• Purification strategy: Ensure your purification approach maintains protein integrity and tetrameric structure

Regular verification of protein expression by Western blotting and activity assays is recommended to monitor consistency.

How can researchers distinguish between glycolytic and non-glycolytic functions of Pfkp in experimental systems?

Distinguishing between the glycolytic and non-glycolytic functions of Pfkp requires multifaceted experimental approaches:

• Site-directed mutagenesis: Generate mutants that specifically affect either glycolytic or non-glycolytic functions. For example, mutations at the catalytic site may affect glycolytic function while potentially preserving protein-protein interactions

• Protein-protein interaction studies: Use co-immunoprecipitation followed by mass spectrometry to identify interacting partners, as demonstrated with the identification of Lin41 as a Pfkp-interacting protein

• Domain mapping experiments: Define regions in Pfkp responsible for specific interactions, similar to the approach used to map interactions between Pfkp and Lin41

• Cellular localization studies: Determine if Pfkp localizes to different cellular compartments when performing glycolytic versus non-glycolytic functions

• Metabolic vs. non-metabolic readouts: Measure both glycolytic parameters (e.g., lactate production) and non-glycolytic outcomes (e.g., stem cell differentiation markers) in parallel experiments with wild-type and mutant Pfkp

This multi-parameter analysis can help delineate which experimental outcomes stem from glycolytic versus non-glycolytic functions.

What emerging roles for Pfkp beyond metabolism warrant further investigation?

Several emerging roles for Pfkp beyond its canonical metabolic function deserve further investigation:

• Stem cell biology: The discovered role of Pfkp in regulating pluripotency and lineage specification in embryonic stem cells represents a significant non-canonical function . Further research could explore:

  • The exact mechanism by which Pfkp-mediated Lin41 phosphorylation affects differentiation pathways

  • The potential role of Pfkp in adult stem cell regulation and tissue regeneration

  • Whether Pfkp functions similarly in induced pluripotent stem cells

• Cancer biology: The finding that PFKP shows prognostic value across multiple cancer types raises questions about:

  • Mechanistic links between Pfkp and cancer progression

  • The role of Pfkp in cancer metabolism reprogramming

  • Potential for Pfkp-targeted therapy approaches

• Immune regulation: PFKP's correlation with PD-L1 suggests unexplored immunomodulatory functions that could impact:

  • Immune cell metabolism and function

  • Tumor immune evasion mechanisms

  • Immunotherapy response prediction

• Mitophagy regulation: The interaction between PFKP and FUNDC1 in mitophagy pathways opens questions about:

  • The evolutionary conservation of this function across species

  • The broader role of Pfkp in cellular quality control mechanisms

  • Potential therapeutic applications in degenerative diseases

What technological advances could enhance Pfkp research methodologies?

Future Pfkp research could benefit from several technological advances:

• Structural biology techniques: High-resolution structures of rat Pfkp in different conformational states would provide deeper insights into its regulation and function

• CRISPR-Cas9 genome editing: Generation of precise knock-in and conditional knockout models could allow tissue-specific and time-controlled Pfkp modulation

• Single-cell metabolomics: This emerging technology could reveal how Pfkp activity varies at the single-cell level, potentially uncovering cell-to-cell variability in metabolic states

• Phosphoproteomics: Comprehensive analysis of proteins phosphorylated by Pfkp could expand our understanding of its non-glycolytic functions

• Spatial transcriptomics and proteomics: These techniques could map Pfkp expression and activity patterns within complex tissues, providing context for its diverse functions

These technological approaches would enhance our ability to understand the multifaceted roles of Pfkp in cellular physiology and pathology.