Recombinant Plasmodium falciparum Flap endonuclease 1 (FEN1), partial

What is Plasmodium falciparum Flap Endonuclease 1 and what role does it play in the parasite lifecycle?

PfFEN-1 is a structure-specific endonuclease that plays a critical role in resolving single-stranded DNA flap intermediates during long patch DNA Base Excision Repair (BER) . Unlike mammalian systems that predominantly use short patch BER for repairing AP (apurinic/apyrimidinic) sites, P. falciparum relies almost exclusively on the long patch BER pathway . This makes FEN-1 particularly crucial for the parasite's DNA repair mechanisms.

The expression of PfFEN-1 has been detected in all erythrocytic stages of the parasite lifecycle (ring, trophozoite, and schizont stages) through Western blot analysis using purified anti-PfFEN-1 antibodies . The consistent expression throughout these stages indicates its essential role in maintaining genomic integrity during parasite development.

How does the structure of PfFEN-1 differ from other eukaryotic FEN1 enzymes?

Protein sequence analysis has revealed significant structural differences between PfFEN-1 and other eukaryotic FEN1 homologs:

• While the N and I domains of PfFEN-1 share homology with FEN-1 from other species, PfFEN-1 possesses an extended C domain with limited homology to other apicomplexan FEN-1s and no homology to eukaryotic FEN-1s .

• PfFEN-1 features a conserved Proliferating Cell Nuclear Antigen (PCNA) binding site at an internal location rather than at the extreme C-terminal position typically observed in FEN-1 from other organisms .

• PfFEN-1 (~75 kDa) is substantially larger than human FEN-1, primarily due to its extended C-terminal domain containing codon repeats and regions of low complexity .

• This extended C-terminus is characteristic of apicomplexan parasites, with PfFEN-1 having one of the largest C domains among all FEN-1 homologs investigated to date .

What are the main challenges in expressing recombinant PfFEN-1 and how can they be overcome?

The expression of recombinant PfFEN-1 presents several significant challenges:

• The extremely high AT content (71.8%) of the native P. falciparum gene sequence makes bacterial expression difficult .

• Initial attempts to express PfFEN-1 in various systems (bacterial, insect, and cell-free) were unsuccessful, necessitating alternative approaches .

These challenges were overcome through codon optimization strategies:

• The gene sequence was optimized to match human codon usage, reducing the AT content from 71.8% to 59.7% to facilitate bacterial expression .

• The optimized gene was synthesized and cloned into the pCAL-n-FLAG expression vector, which incorporates both calmodulin-binding peptide (CBP) and FLAG tag sequences at the 5′ end of the PfFEN-1 gene .

• The construct was transformed into BLR(DE3)pLysS E. coli cells, resulting in successful expression of the recombinant protein .

What is the recommended purification protocol for obtaining high-purity recombinant PfFEN-1?

Based on published research, the optimal purification protocol for recombinant PfFEN-1 involves a two-step chromatography approach:

• Initial purification using an SP (sulfopropyl) cation exchange column, which yields a relatively impure protein fraction .

• Secondary purification via a CAR (calmodulin affinity resin) column, which results in >95% pure PfFEN-1 .

For the truncated version lacking the terminal 250 amino acids (PfFEN-1ΔC), the recommended purification protocol differs:

• Primary purification using a Ni-NTA (nickel-nitrilotriacetic acid) column .

• Secondary purification using an SP column chromatography step to achieve >95% purity .

The identity and purity of the purified proteins should be confirmed through Western blotting using appropriate antibodies .

How can the endonuclease and exonuclease activities of recombinant PfFEN-1 be measured in vitro?

The enzymatic activities of recombinant PfFEN-1 can be assessed using the following in vitro assays:

For endonuclease activity:

• Add 10.0 nM Flap34 endo substrate to FEN-1 reaction buffer

• Add empirically determined amounts of FEN-1 (1.5 nM PfFEN-1, 0.005 nM PfFEN-1ΔC, or 7.0 nM PyFEN-1)

• Incubate samples at 37°C for 30 minutes

• Analyze the reaction products

For exonuclease activity:

• Add 10.0 nM Flap34 exo substrate to FEN-1 reaction buffer

• Add empirically determined amounts of FEN-1 (15.0 nM PfFEN-1, 0.5 nM PfFEN-1ΔC, or 7.0 nM PyFEN-1)

• Incubate samples at 37°C for 30 minutes

• Analyze the reaction products

These assays have confirmed that PfFEN-1 possesses both DNA structure-specific flap endonuclease and 5′→3′ exonuclease activities, similar to FEN-1 homologs from other species, with endonuclease activity being more robust than exonuclease activity .

What factors influence the enzymatic activity of PfFEN-1?

Several factors have been identified that significantly affect PfFEN-1 enzymatic activity:

• Divalent metal ions: Endonuclease activity is stimulated by Mg²⁺ or Mn²⁺, indicating a requirement for these divalent cations in the catalytic mechanism .

• Monovalent ions: Endonuclease activity is inhibited by monovalent ions at concentrations exceeding 20.0 mM .

• C-terminal domain: The extended C-terminal domain appears to negatively regulate enzymatic activity. The truncated mutant PfFEN-1ΔC demonstrated approximately 130-fold greater endonuclease activity (kcat = 1.2×10⁻¹) compared to full-length PfFEN-1 (kcat = 9.1×10⁻⁴) and approximately 240-fold greater activity than PyFEN-1 (kcat = 4.9×10⁻⁴) in vitro .

• Low complexity regions: The C-terminal domain contains low complexity regions rich in lysine and asparagine residues, which may play a critical but not fully understood role in regulating the biochemical activity of apicomplexan FEN-1s .

How does the catalytic efficiency of PfFEN-1 compare with its truncated version and other FEN1 homologs?

The catalytic parameters of different FEN1 variants show remarkable differences as summarized in the table below:

This data clearly demonstrates that removal of the C-terminal domain significantly enhances the catalytic efficiency of PfFEN-1, suggesting this domain plays a regulatory role in modulating enzymatic activity . The truncated version more closely resembles other eukaryotic FEN-1 homologs in both size and activity levels.

How can PfFEN-1 be used to study the unique DNA repair mechanisms in Plasmodium parasites?

PfFEN-1 serves as a valuable tool for investigating the distinctive DNA repair mechanisms in Plasmodium species:

• Reconstitution of long patch BER pathway: Studies have shown that PfFEN-1 can generate a nicked DNA substrate that is subsequently ligated by recombinant P. falciparum DNA Ligase I (PfLigI), enabling in vitro reconstitution of key steps in the long patch BER pathway .

• Comparative analysis with other organisms: P. falciparum uniquely relies on long patch BER rather than short patch BER for DNA repair. The P. falciparum genome lacks homologs of proteins essential for short patch BER (DNA polymerase β, XRCC1, and DNA Ligase III) while possessing proteins required for long patch BER (DNA glycosylases, AP endonucleases, DNA Polymerase δ, RFC, RPA, PCNA, FEN-1, and DNA Ligase I) .

• Adaptation to AT-rich genome: With an extremely AT-rich genome, P. falciparum may require specific adaptations in its DNA repair enzymes. The unique structural features of PfFEN-1 might represent such adaptations, providing insights into how the parasite maintains genomic integrity .

What is the significance of the extended C-terminal domain in PfFEN-1 function?

The extended C-terminal domain of PfFEN-1 represents one of its most distinctive features with significant implications for enzyme function:

• Activity regulation: The C-terminal domain appears to negatively regulate enzymatic activity, as evidenced by the dramatically increased catalytic efficiency of the truncated PfFEN-1ΔC variant .

• Evolutionary conservation: All apicomplexan parasites, including all Plasmodium species, contain extended C-domains of varying lengths, suggesting an important evolutionary adaptation specific to this group of organisms .

• Structural characteristics: The C-terminal domain is primarily composed of low complexity regions with repeated amino acids, typically characterized by an abundance of lysine and asparagine residues .

• Functional implications: While the precise function of these low complexity regions is not fully understood, they have been demonstrated to be essential for the enzymatic activity of some apicomplexan enzymes and may play a critical role in protein-protein interactions or other regulatory functions .

• Potential target for intervention: The unique nature of this domain makes it a potential target for selective inhibition of PfFEN-1 without affecting human FEN1.

How does FEN1 inhibition affect cellular processes and what are the implications for drug development?

FEN1 inhibition has significant effects on cellular processes, particularly in cells with defective DNA repair mechanisms:

• Disruption of DNA replication: Treatment with FEN1 inhibitors (such as compound C8) reduces BrdU incorporation in cells, indicating inhibition of DNA synthesis .

• Cell cycle perturbation: FEN1 inhibition leads to changes in cell cycle distribution, including increases in G2/M fraction and varying effects on G1 and sub-G1 populations depending on the cell line .

• DNA damage induction: FEN1 inhibition induces DNA damage response, as evidenced by the accumulation of histone γH2AX .

• Differential recovery capacity: While both sensitive and resistant cell lines show DNA damage response following FEN1 inhibition, sensitive cell lines are unable to recover and replicate DNA even after inhibitor removal .

These cellular effects demonstrate the potential of FEN1 inhibitors as therapeutics, particularly for targeting cells with specific DNA repair deficiencies.

What features of PfFEN-1 could be exploited for the development of selective antimalarial agents?

Several unique characteristics of PfFEN-1 present opportunities for developing selective antimalarial agents:

• Extended C-terminal domain: The presence of an extended C-terminal domain not found in human FEN1 provides a potential target for selective inhibition .

• Internal PCNA binding site: The unusually positioned PCNA binding site in PfFEN-1 might allow for the development of inhibitors that specifically disrupt this interaction without affecting human FEN1-PCNA binding .

• Regulatory mechanisms: Understanding how the C-terminal domain regulates PfFEN-1 activity could guide the design of allosteric inhibitors that specifically modulate parasite enzyme function .

• Essential role in parasite DNA repair: P. falciparum's reliance on long patch BER, with FEN-1 playing a critical role, suggests that targeting this enzyme could be particularly effective against the parasite while potentially sparing human cells that possess alternative repair pathways .

• Precedent from cancer research: The successful development of FEN1 inhibitors for targeting cancers with homologous recombination defects provides a potential model for antimalarial drug development focused on PfFEN-1 .

What are the key considerations when designing experiments to investigate PfFEN-1 function in vitro?

When designing experiments to investigate PfFEN-1 function, researchers should consider:

• Protein expression optimization: Given the challenges in expressing PfFEN-1, codon optimization is crucial for successful production of recombinant protein .

• Purification strategy: A multi-step purification approach is necessary to obtain highly pure protein, with specific protocols differing between full-length and truncated variants .

• Enzyme concentration: Due to differences in activity levels, optimal enzyme concentrations vary significantly between PfFEN-1 (1.5 nM for endonuclease assays), PfFEN-1ΔC (0.005 nM), and PyFEN-1 (7.0 nM) .

• Buffer conditions: Reaction buffers should be optimized considering that endonuclease activity is stimulated by Mg²⁺ or Mn²⁺ and inhibited by monovalent ions at concentrations exceeding 20.0 mM .

• Substrate design: Structure-specific substrates that mimic the DNA flap structures encountered in vivo should be used for accurate assessment of enzymatic activity .

• Activity comparison: When comparing different variants, catalytic parameters (kcat) should be determined under standardized conditions to enable meaningful comparisons .

How can researchers address the challenges in studying the structure-function relationship of PfFEN-1's extended C-terminal domain?

The extended C-terminal domain of PfFEN-1 presents unique challenges for structure-function studies that can be addressed through:

• Systematic truncation analysis: Creating a series of truncation mutants with varying lengths of the C-terminal domain can help identify specific regions critical for activity regulation .

• Chimeric protein construction: Swapping the C-terminal domains between PfFEN-1 and FEN1 from other species can help determine the domain's specific roles.

• Site-directed mutagenesis: Targeting conserved residues within the low complexity regions can help identify specific amino acids essential for function.

• Structural studies: While challenging due to the disordered nature of the C-terminal domain, techniques such as small-angle X-ray scattering (SAXS) or hydrogen-deuterium exchange mass spectrometry could provide insights into the domain's structural dynamics.

• Protein-protein interaction studies: Investigating potential interaction partners of the C-terminal domain may reveal its functional significance in the context of DNA repair complexes.

• Comparative analysis: Examining the C-terminal domains of FEN-1 from different Plasmodium species and other apicomplexan parasites could reveal evolutionarily conserved features that are functionally important.