Recombinant Candida glabrata Serine/threonine-protein phosphatase 4 catalytic subunit (PPH3)

What is PPH3 and what is its significance in Candida glabrata?

PPH3 (Serine/threonine-protein phosphatase 4 catalytic subunit) is a member of the PPP family of protein phosphatases in Candida glabrata. As a catalytic subunit of the protein phosphatase 4 complex, PPH3 plays critical roles in various cellular processes, particularly in DNA damage response pathways. The PPH3 protein in C. glabrata shares functional homology with similar phosphatases in other fungi such as Candida albicans and Saccharomyces cerevisiae, where they regulate stress responses, cell cycle progression, and morphogenesis. Based on studies in C. albicans, PPH3 likely forms a complex with its regulatory subunit PSY2 to perform its cellular functions .

What expression systems are most effective for producing recombinant C. glabrata PPH3?

For recombinant PPH3 expression, E. coli-based systems have been successfully employed, as indicated by the product information in the search results. For optimal expression, consider the following methodological approach:

• Vector selection: Use vectors containing strong inducible promoters (T7, tac) suitable for toxic proteins.

• Host strains: BL21(DE3) or Rosetta strains can be used to address potential codon bias issues.

• Expression conditions: Induction at lower temperatures (16-20°C) often improves solubility of phosphatases.

• Co-expression strategies: Consider co-expressing PPH3 with its regulatory subunit PSY2 to improve folding and activity.

When working with fungal proteins in bacterial systems, codon optimization may be necessary to overcome expression barriers. Additionally, including appropriate affinity tags (His, GST) facilitates purification while maintaining enzymatic activity .

How can researchers effectively measure PPH3 phosphatase activity?

To assess PPH3 phosphatase activity, multiple complementary approaches can be employed:

In vitro phosphatase assays:

• pNPP hydrolysis assay: Using para-nitrophenylphosphate as a colorimetric substrate.

• Malachite green assay: Measuring released phosphate from phosphopeptide substrates.

• Specific substrate assays: Using phosphorylated Rad53 isolated from MMS-treated cells as a physiologically relevant substrate.

Cellular functional assays:

• Cell recovery experiments: Measuring the recovery of C. glabrata cells from MMS-induced DNA damage in wild-type vs. pph3Δ strains.

• Rad53 phosphorylation status: Western blotting with phospho-specific antibodies to monitor Rad53 phosphorylation levels.

• Cell cycle analysis: Flow cytometry to analyze the cell cycle progression following DNA damage in the presence/absence of PPH3.

When working with recombinant PPH3, it's critical to include metal cofactors (Mn²⁺ or Mg²⁺) in reaction buffers and use physiological pH conditions (pH 7.0-7.5) .

What is the specific role of PPH3 in DNA damage response pathways?

Based on studies in C. albicans, PPH3 plays a crucial role in the recovery from DNA damage, particularly that induced by methyl methanesulfonate (MMS). The primary mechanism involves:

• Rad53 dephosphorylation: PPH3, in complex with PSY2, dephosphorylates the checkpoint kinase Rad53, which is essential for cell cycle restart after DNA damage repair.

• Damage-specific function: Interestingly, PPH3 appears to be specifically required for recovery from MMS-induced DNA damage but not from hydroxyurea (HU)-induced replication stress. This suggests distinct phosphorylation patterns and recovery pathways for different types of genotoxic stress.

• Morphological regulation: In C. albicans, PPH3 deletion results in enhanced and prolonged filamentous growth in response to MMS treatment. This suggests that PPH3 also regulates morphogenesis in response to DNA damage.

• Transcriptional regulation: PPH3 influences the expression of genes controlled by the MBF transcription factor, which regulates DNA replication and repair genes, and also affects the expression of hypha-specific genes in C. albicans .

How do PPH3 and Rad53 interact in the context of checkpoint recovery?

The interaction between PPH3 and Rad53 represents a critical regulatory mechanism in DNA damage checkpoint recovery:

• Specific dephosphorylation sites: Research in C. albicans has identified S461 and S545 on Rad53 as potential sites for PPH3/PSY2-mediated dephosphorylation that are specifically involved in cellular responses to MMS.

• Phosphorylation dynamics: Following MMS treatment, Rad53 becomes hyperphosphorylated, activating the DNA damage checkpoint. In wild-type cells, PPH3/PSY2 dephosphorylates Rad53 during recovery, allowing cell cycle restart. In pph3Δ or psy2Δ mutants, Rad53 remains hyperphosphorylated even after MMS removal.

• Mutant phenotypes: Mutation studies using phosphomimetic (S→D) and phosphodeficient (S→A) versions of Rad53 at positions S461 and S545 have demonstrated that these phosphorylation sites are specifically involved in MMS response but not HU response.

This specific dephosphorylation pattern illustrates how the same protein (Rad53) can be differentially regulated depending on the type of DNA damage, with PPH3 playing a crucial role in this selective response .

How does PPH3 function differ between MMS and HU treatment in Candida species?

The differential role of PPH3 in response to MMS versus HU represents a fascinating example of context-dependent phosphatase function:

This distinction suggests that different DNA-damaging agents activate distinct signaling pathways that involve different phosphatases for checkpoint recovery. While PPH3/PSY2 is critical for recovery from MMS-induced damage, other phosphatases (possibly Glc7 as suggested by studies in S. cerevisiae) may be responsible for recovery from HU-induced replication stress .

How can CRISPR-Cas9 be applied for manipulating PPH3 in Candida glabrata?

The CRISPR-Cas9 system offers a powerful alternative for genome engineering in C. glabrata. Based on the search results describing CRISPR-Cas9 implementation in C. glabrata, researchers can apply the following methodology:

• Development of a CRISPR-Cas9 expression system:

  • Generate a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system.

  • Design guide RNAs targeting PPH3 using an online program that facilitates the selection of efficient guide RNAs.

• Implementation steps:

  • Transform cells with a construct containing the guide RNA targeting PPH3 and a repair template (if generating specific mutations rather than deletions).

  • Select transformants and screen using the Surveyor technique to identify mutations.

  • Validate mutations by sequencing.

• Validation of phenotypes:

  • Test mutant strains for altered responses to DNA-damaging agents, particularly MMS sensitivity.

  • Assess changes in cell morphology, Rad53 phosphorylation status, and cell cycle progression.

  • Evaluate virulence using appropriate infection models, such as Drosophila melanogaster or Galleria mellonella.

This approach allows for precise genetic manipulation and can be used to introduce specific point mutations in PPH3 to study structure-function relationships .

How does PPH3 function in the broader context of phosphatase networks in Candida glabrata?

PPH3 operates within a complex network of protein phosphatases that collectively regulate cellular responses to stress and environmental changes:

• Phosphatase redundancy and specialization:

  • Studies in S. cerevisiae and C. albicans suggest that while some phosphatases show functional overlap, others have specialized roles in specific stress responses.

  • For example, while PPH3 is crucial for recovery from MMS-induced damage, Glc7 (PP1) may be more important for recovery from HU-induced replication stress.

  • The dual phosphatase system (PPH3 and PTC2) shows both independent and redundant functions in response to genotoxic stress, with the pph3Δ ptc2Δ double mutant exhibiting greater sensitivity than either single mutant.

• Phosphatase-kinase balance:

  • PPH3 functions as a counterbalance to checkpoint kinases, particularly in the DNA damage response pathway.

  • This balance is critical for appropriate activation and deactivation of checkpoint mechanisms.

• Substrate specificity:

  • Different phosphatases target specific phosphorylation sites even on the same protein (as seen with Rad53), allowing for nuanced regulation of cellular responses.

  • This specificity is often mediated by regulatory subunits (like PSY2 for PPH3) that direct the catalytic subunit to specific subcellular locations or substrates.

Understanding this network is essential for developing comprehensive models of stress response regulation in pathogenic fungi .

What is the relationship between PPH3 and antifungal resistance mechanisms?

While the direct role of PPH3 in antifungal resistance is not extensively documented in the provided search results, several connections can be inferred:

• Stress response pathways:

  • PPH3's role in DNA damage response links it to general stress adaptation mechanisms that may contribute to antifungal tolerance.

  • The persistent filamentous growth observed in pph3Δ mutants after MMS treatment suggests that PPH3 regulation may influence morphological transitions that are known to affect antifungal susceptibility.

• Connection to calcineurin pathway:

  • The search results indicate that calcineurin, another important phosphatase, is required for transcriptional activation by Pdr1, a transcription factor involved in fluconazole resistance in C. glabrata.

  • Given that phosphatases often function in interconnected networks, PPH3 might indirectly influence antifungal resistance pathways regulated by calcineurin.

• DNA damage and drug resistance:

  • Some antifungals induce DNA damage or oxidative stress that activates DNA damage response pathways.

  • PPH3's role in regulating recovery from such damage suggests potential involvement in cellular responses to antifungal drugs that operate through these mechanisms.

Research examining the phosphorylation status of key resistance determinants in wild-type versus pph3Δ strains during antifungal treatment would provide valuable insights into these potential connections .

How can phosphoproteomic approaches be used to identify novel PPH3 substrates?

Phosphoproteomics offers powerful strategies for identifying PPH3 substrates in C. glabrata:

• Comparative phosphoproteomics workflow:

  • Culture wild-type and pph3Δ mutant strains under normal conditions and after MMS treatment.

  • Extract and digest proteins, followed by enrichment of phosphopeptides using titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC).

  • Analyze enriched phosphopeptides by LC-MS/MS.

  • Identify peptides showing increased phosphorylation in the pph3Δ strain compared to wild-type, particularly after MMS treatment.

• In vitro validation approaches:

  • Express and purify recombinant PPH3/PSY2 complex.

  • Generate phosphorylated candidate substrates (either synthetically or by in vitro kinase reactions).

  • Perform dephosphorylation assays with purified PPH3/PSY2.

  • Monitor dephosphorylation by mass spectrometry or phospho-specific antibodies.

• Integration with interactome data:

  • Perform immunoprecipitation of tagged PPH3 followed by mass spectrometry to identify interacting proteins.

  • Cross-reference interactome data with phosphoproteome data to prioritize likely direct substrates.

  • Validate physical interactions using techniques such as yeast two-hybrid or bimolecular fluorescence complementation.

This multifaceted approach would enable the identification of the PPH3 substrate network beyond the currently known target Rad53, providing a more comprehensive understanding of its cellular functions .

What is the significance of PPH3 in C. glabrata virulence and pathogenesis?

While the direct link between PPH3 and virulence in C. glabrata is not extensively documented in the provided search results, several lines of evidence suggest potential connections:

• Stress adaptation and survival:

  • PPH3's role in recovery from DNA damage suggests it may contribute to survival in host environments where oxidative stress from immune cells can cause DNA damage.

  • The ability to recover from stress is a key virulence attribute for successful pathogens.

• Morphological transitions:

  • In C. albicans, PPH3 regulates filamentous growth in response to MMS, suggesting a role in morphological plasticity.

  • Although C. glabrata does not form true hyphae like C. albicans, morphological transitions still contribute to its pathogenesis.

• Comparative analysis with other virulence factors:

  • The search results indicate that other phosphatases, like calcineurin, are involved in virulence and drug resistance.

  • The multidrug transporter CgDtr1 was identified as a determinant of C. glabrata virulence in the Galleria mellonella infection model, illustrating how proteins involved in stress responses can impact virulence.

A systematic evaluation of pph3Δ mutants in appropriate infection models would be necessary to definitively establish its role in virulence .

How do PPH3 functions differ between Candida glabrata and other Candida species?

Understanding the species-specific functions of PPH3 is important for developing targeted antifungal strategies:

These differences highlight the importance of species-specific studies rather than assuming conserved functions across all Candida species, especially given C. glabrata's closer phylogenetic relationship to S. cerevisiae than to C. albicans .

What are the most promising research directions for understanding PPH3 function in Candida glabrata?

Based on current knowledge and gaps identified in the search results, several high-priority research areas emerge:

• Comprehensive substrate identification:

  • Application of phosphoproteomic approaches to identify the full range of PPH3 substrates beyond Rad53.

  • Investigation of substrate specificity differences between PPH3 in C. glabrata versus other fungal species.

• Structural biology approaches:

  • Determination of the crystal structure of PPH3 alone and in complex with PSY2 and substrates.

  • Structure-guided design of specific inhibitors as potential antifungal agents.

• Systems biology integration:

  • Mapping the position of PPH3 within the broader phosphorylation/dephosphorylation networks in C. glabrata.

  • Identification of cross-talk between PPH3 and other stress response pathways, including those involved in antifungal resistance.

• Translational applications:

  • Evaluation of PPH3 as a potential antifungal target, particularly in combination therapies targeting multiple phosphatases.

  • Development of diagnostic approaches based on phosphorylation signatures indicative of drug resistance.