Recombinant Human Furin (FURIN)

What is recombinant human furin and how is it functionally characterized?

Recombinant human furin is a proprotein convertase enzyme typically produced using expression systems such as Chinese Hamster Ovary (CHO) cells. Commercial preparations often contain the catalytic domain (Asp108-Glu715) with a C-terminal His-tag to facilitate purification . Furin functions by cleaving inactive protein precursors in the secretory pathway, thereby controlling the activation of diverse types of proteins including extracellular matrix proteins, signaling peptides, hormones, growth factors, serum proteins, transmembrane receptors, ion channels, bacterial toxins, and viral fusion peptides .

Functional characterization typically involves fluorogenic substrate assays. A standard protocol includes:

• Diluting rhFurin to 4 μg/mL in appropriate assay buffer

• Using p-Glu-Arg-Thr-Lys-Arg-AMC as a fluorogenic substrate (diluted to 100 μM)

• Measuring activity at excitation/emission wavelengths of 380/460 nm

• Monitoring reaction kinetics for 5 minutes using a fluorescent plate reader

What structural features define furin's active site and substrate recognition?

Furin's active site contains a high negative-charge density that plays a crucial role in substrate recognition. Crystal structure analysis at 1.8-Å resolution has revealed substantial differences between the unliganded state and inhibitor-bound forms, particularly in the active-site residues and substrate-binding cleft .

The substrate recognition motif typically spans 20 residues, with the core consensus sequence R-X-K/R-R↓ at the cleavage site (where ↓ indicates the cleavage position). The P1 position strongly prefers arginine, while the P2 position accepts basic residues (Lys/Arg), and the P4 position also shows preference for basic residues. The three-dimensional configuration of these residues within the substrate is critical for proper orientation in furin's active site to enable enzymatic cleavage .

How do expression systems affect recombinant human furin production?

Expression systems significantly impact both the yield and quality of recombinant human furin. CHO cells are commonly used due to their ability to perform appropriate post-translational modifications. When expressing furin at high levels (up to 120 μg/mL × day), certain processing steps can become impaired, affecting the functionality of the final product .

Key considerations for expression systems include:

• Selection of cell lines with appropriate post-translational modification capabilities

• Optimization of culture conditions including vitamin K availability for γ-carboxylation

• Monitoring of propeptide removal and chain processing

• Assessment of functional activity relative to expression level

Research has shown that even at high expression levels that impair some processing steps, up to 25% of recombinant human furin can retain activity, which represents the highest functional activity reported for vitamin K-dependent proteins at such expression levels .

How can computational tools predict furin cleavage sites in target proteins?

Computational prediction of furin cleavage sites has evolved to include sophisticated hybrid methods. The PiTou prediction tool combines a hidden Markov model with biological knowledge-based cumulative probability score functions to identify potential furin cleavage sites with high sensitivity (96.9%) and specificity (97.3%) .

PiTou scores range from 0.01 to 0.99, with scores above 0.7 accurately predicting efficient furin cleavage. The scores correlate with biological attributes including:

• Binding strength between furin and substrate

• Solvent accessibility of the cleavage site

• Conformational compatibility with the furin active site

For effective utilization in research:

• Analyze your protein sequence using the PiTou algorithm

• Consider scores in context of subcellular localization and competing modifications

• Validate predictions experimentally using recombinant furin and synthetic peptides

• Interpret results within cellular contexts including subcellular localization and potential interference by other dynamic protein modifications

What are the molecular determinants that influence furin cleavage efficiency?

Despite containing predicted furin cleavage motifs, some proteins resist furin cleavage due to subtle structural constraints. Research on coronavirus spike proteins has revealed several key molecular determinants affecting cleavage efficiency:

• The identity of the residue in the P2 position critically influences cleavage. For example, a histidine residue in this position in MHV-A59 spike protein fails to properly orient the sidechain of His194 in furin's catalytic triad, making it incompatible with cleavage initiation despite a high predictive score .

• The P1 position typically requires arginine, but research has shown that Ser/Thr residues in this position (as found in MHV-2 and MHV-S spike proteins) distort the conformation of the furin active site, explaining altered cleavage patterns .

• The region downstream of the cleavage site (P1'-P6') also influences cleavage efficiency. Enrichment of hydrophobic residues in this region has been shown to interfere with furin cleavage efficiency .

• Acidic anchor residues (like glutamate) at P5/P6 positions can significantly inhibit furin catalysis without affecting binding .

Molecular dynamics simulations provide valuable insights into how potential substrates bind to furin's active site and whether the resulting complex is suitable for initiating enzymatic cleavage .

What strategies can be employed to engineer furin inhibition for therapeutic applications?

Furin inhibition has emerged as a potential therapeutic strategy, particularly for viral infections where furin-mediated proteolytic activation is critical. Advanced engineering approaches include:

• Antibody-based targeted strategies: The "FuG1" approach combines an anti-spike IgG1 with an engineered Fc-extended peptide capable of competitively inhibiting the furin substrate-binding pocket. This dual functionality allows simultaneous targeting of viral proteins and inhibition of furin-mediated cleavage .

• Competitive substrate design: Engineering peptides with specific modifications can create competitive inhibitors that bind furin without being cleaved. Key design principles include:

  • Incorporating nonpolar hydrophobic residues in the P1′ to P6′ region

  • Adding side-chained cysteine (GGCPGS) in strategic positions

  • Inserting acidic anchor glutamate (E) at P5/P6 positions

• Optimizing inhibitor binding kinetics: While the dissociation constant (KD) and association rate (KON) of inhibitors like FuG1 may be lower compared to natural substrates, a reduced dissociation rate (KOFF) can provide higher competitive furin occupancy time with increased antibody concentration .

Experimental validation requires careful assessment of furin binding versus cleavage using techniques like SDS-PAGE analysis of potential substrate proteins incubated with recombinant furin .

How does furin expression correlate with metabolic disorders and disease outcomes?

Plasma furin levels show significant associations with metabolic disorders and mortality risk. A population-based cohort study revealed that individuals with high plasma furin concentrations display:

Statistical analysis demonstrated significant correlations between furin levels and:

• Body mass index (β = 0.31, P < 0.001)

• Blood pressure

• Plasma glucose concentration

• Insulin levels

• LDL and HDL cholesterol levels

Importantly, individuals in the highest quartile of furin concentration showed elevated risk of developing diabetes mellitus and higher rates of premature mortality. These findings suggest that furin may serve as a biomarker for metabolic disease risk and could potentially represent a therapeutic target .

How should researchers optimize furin activity assays for different experimental contexts?

Optimization of furin activity assays requires attention to several key parameters:

• Buffer composition:

  • Use 100 mM HEPES, pH 7.5

  • Include 0.5% Triton X-100

  • Add 1 mM CaCl₂

  • Include 1 mM 2-mercaptoethanol

• Substrate selection:

  • Standard substrate: p-Glu-Arg-Thr-Lys-Arg-AMC

  • Ensure substrate concentration falls within linear range (typically 50-200 μM)

  • Consider testing substrate analogs for specific applications

• Enzyme concentration:

  • Standard concentration: 4 μg/mL

  • Adjust based on expected activity and detection limits

  • Include appropriate negative controls to account for background

• Detection parameters:

  • Excitation/emission: 380/460 nm

  • Kinetic mode reading over 5 minutes

  • Black 96-well plates to minimize background

When adapting assays to study potential inhibitors or modified substrates, include appropriate controls and consider performing dose-response analyses to accurately quantify effects on furin activity.

What considerations are important when designing experiments to study furin-dependent protein processing?

When investigating furin-dependent protein processing, several experimental design considerations are critical:

• Cell model selection:

  • CHO cells are commonly used for expression studies

  • Consider using furin-deficient cell lines to demonstrate furin-dependency

  • Expression in CHO cells lacking endoprotease furin has revealed that some processing steps (e.g., propeptide removal in factor X) can still occur, suggesting involvement of different proteases

• Processing analysis:

  • Monitor multiple processing events separately (e.g., propeptide removal versus chain processing)

  • Characterize processing intermediates using techniques like Western blotting and mass spectrometry

  • Consider temporal dynamics of processing events

• Structural considerations:

  • Evaluate the impact of substrate sequence variations around the cleavage site

  • Consider three-dimensional structural constraints that may affect accessibility

  • Use molecular dynamics simulations to predict binding modes and cleavage efficiency

• Functional validation:

  • Correlate processing state with functional activity of the target protein

  • Include both positive and negative controls for furin cleavage

  • Consider the impact of warfarin treatment on vitamin K-dependent proteins

Research on recombinant human factor X has demonstrated that despite impaired processing at high expression levels, up to 25% of the protein can remain functionally active, highlighting the complexity of furin-dependent processing systems .

How can researchers effectively target furin for viral inhibition studies?

Designing experiments to evaluate furin inhibition in viral contexts requires careful consideration of several factors:

• Target selection:

  • Identify specific viral proteins dependent on furin processing

  • For coronaviruses, the S1/S2 site of the spike protein is a primary target

  • Consider strain-specific variations in cleavage site sequences

• Inhibition strategy design:

  • Competitive inhibitors based on substrate-like peptides

  • Antibody-based targeted approaches (e.g., FuG1)

  • Small molecule inhibitors targeting the furin active site

• Validation methodology:

  • In vitro cleavage assays using recombinant furin and viral protein substrates

  • Cell-based viral infection assays in the presence of inhibitors

  • Binding kinetics studies to assess KD, KON, and KOFF

• Optimization parameters:

  • PiTou score optimization for competitive peptides (scores near 0.5 may bind without being cleaved)

  • Enrichment of hydrophobic residues in P1' to P6' regions to interfere with cleavage efficiency

  • Addition of acidic anchor residues at P5/P6 positions

This approach has been successfully demonstrated with the FuG1 inhibitor design, which combines an anti-spike IgG1 with a furin-binding peptide engineered to have a PiTou score of 0.504 - high enough for binding but below the optimal cleavage threshold .

How should researchers interpret disparities between predicted and actual furin cleavage patterns?

Discrepancies between computational predictions and experimental observations of furin cleavage require careful analysis:

• Sequence vs. structure considerations:

  • High PiTou scores (>0.7) generally predict efficient cleavage, but exceptions exist

  • MHV-A59 spike protein exemplifies how a high prediction score may not translate to actual cleavage due to structural constraints

  • Three-dimensional structural requirements of the furin active site configuration determine which bound peptides undergo cleavage

• Contextual factors to evaluate:

  • Accessibility of the cleavage site in the folded protein

  • Local structural elements that may impede enzyme-substrate interaction

  • Post-translational modifications affecting recognition

  • Subcellular localization relative to furin distribution

• Investigation approaches:

  • Molecular dynamics simulations to visualize substrate binding modes

  • Mutagenesis studies targeting specific residues around the cleavage site

  • Analysis of processing intermediates to identify rate-limiting steps

The case of MHV-A59 spike protein demonstrates that a histidine residue in the P2 position can fail to properly orient the catalytic triad of furin despite strong sequence-based prediction scores, explaining why cleavage doesn't occur as predicted .

What insights do plasma furin levels provide for metabolic disease research?

Plasma furin measurements offer valuable insights for metabolic disease research, requiring careful interpretation:

When analyzing furin data in metabolic contexts:

• Consider demographic confounders:

  • Age shows strong positive correlation with furin levels

  • Sex distribution varies across furin quartiles

  • Adjust analyses appropriately for these factors

• Evaluate mechanistic links:

  • Furin activates various growth factors and hormones involved in metabolism

  • Processing of insulin receptor and nutrient transporters may be affected

  • Role in adipokine processing may influence systemic inflammation

• Translational potential:

  • Furin levels may serve as biomarkers for metabolic disease risk

  • Therapeutic targeting of furin might address multiple pathways simultaneously

  • Longitudinal studies needed to establish causality versus correlation

These findings suggest that individuals with elevated plasma furin display a pronounced dysmetabolic phenotype and face increased risks of diabetes mellitus and premature mortality, highlighting furin's potential importance in metabolic disease pathophysiology .

What emerging techniques could advance our understanding of furin substrate specificity?

Several cutting-edge approaches show promise for deepening our understanding of furin substrate specificity:

• Cryo-EM analysis of furin-substrate complexes:

  • Capture dynamic interactions during catalytic processing

  • Visualize conformational changes upon substrate binding

  • Reveal structural determinants beyond primary sequence that influence cleavage

• Machine learning approaches:

  • Integrate diverse datasets beyond sequence information

  • Incorporate structural data, solvent accessibility, and post-translational modifications

  • Develop more accurate predictive models for complex substrates

• Proteome-wide identification of furin substrates:

  • Combine next-generation sequencing with computational prediction tools

  • Apply CRISPR-based furin knockout systems to identify differentially processed proteins

  • Develop specialized proteomics approaches to identify furin cleavage products

• Structure-guided engineering:

  • Design highly specific furin inhibitors based on detailed structural knowledge

  • Create substrate analogs with tailored processing kinetics

  • Develop furin variants with modified substrate specificity

These approaches could help elucidate the molecular mechanisms underlying furin cleavage-associated human diseases and inform the development of targeted therapeutic strategies .

How might furin-targeted therapeutics evolve for viral and metabolic diseases?

Furin-targeted therapeutic approaches show considerable promise for both viral infections and metabolic disorders:

• Viral infection applications:

  • Bispecific molecules combining viral targeting with furin inhibition (e.g., FuG1 approach)

  • Structure-guided design of highly specific small molecule inhibitors

  • Prophylactic approaches for high-risk populations

• Metabolic disease applications:

  • Tissue-specific furin inhibition to minimize systemic effects

  • Modulation rather than complete inhibition of furin activity

  • Combination approaches targeting multiple proteases involved in metabolic regulation

• Delivery challenges to address:

  • Targeting inhibitors to relevant cellular compartments

  • Achieving appropriate tissue distribution

  • Minimizing off-target effects due to furin's diverse physiological roles

• Therapeutic design considerations:

  • Competitive inhibitors with PiTou scores near 0.5 for binding without cleavage

  • Incorporation of hydrophobic residues in the P1' to P6' region to reduce cleavage efficiency

  • Strategic placement of acidic residues to inhibit catalysis without affecting binding