Recombinant Candida glabrata 40S ribosomal protein S23 (RPS23)

What is the basic structure and function of RPS23 in Candida glabrata?

RPS23 is a critical component of the 40S ribosomal subunit in Candida glabrata, belonging to the S12P family of ribosomal proteins. Like its homologs in other species, C. glabrata RPS23 is located in the cytoplasm and plays an essential role in protein synthesis machinery . The protein shares significant amino acid sequence similarity with Saccharomyces cerevisiae ribosomal protein S28, reflecting evolutionary conservation among fungal species .

How conserved is RPS23 across fungal species compared to human RPS23?

RPS23 demonstrates remarkable evolutionary conservation across fungal species and even between fungi and humans, particularly within functional domains involved in ribosome assembly and function. Comparative sequence analysis reveals that:

What are the typical expression patterns of RPS23 in different growth phases of Candida glabrata?

Interestingly, research on related pathways suggests that under hypoxic conditions, which C. glabrata may encounter during host colonization, expression patterns may be modulated by transcription factors such as SrbA orthologs . This adaptation allows the pathogen to adjust its translation apparatus according to the microenvironment.

What are the optimal methods for expressing recombinant C. glabrata RPS23?

The expression of recombinant C. glabrata RPS23 presents several technical challenges that require specific methodological approaches:

For structural and functional studies, a common approach involves:

• Gene synthesis with codon optimization for the selected expression system

• Cloning into a vector with an N-terminal His6-tag and a precision protease cleavage site

• Expression in E. coli at reduced temperatures (18°C) after IPTG induction

• Purification using Ni-NTA affinity chromatography followed by size exclusion chromatography

This approach typically yields 5-10 mg of purified protein per liter of culture, sufficient for most biochemical and structural analyses.

What analytical techniques are most effective for studying RPS23 structure and interactions?

Multiple complementary techniques are recommended for comprehensive analysis of C. glabrata RPS23:

For protein-protein interaction studies, a combination of cross-linking followed by mass spectrometry has proven particularly valuable. This approach has successfully identified interaction hotspots between ribosomal proteins and potential regulatory factors, similar to the methodologies used to study SrbA interactions in other fungal systems .

How does RPS23 contribute to Candida glabrata pathogenicity and virulence?

The contribution of RPS23 to C. glabrata pathogenicity appears to be multifaceted:

• Translational adaptation: RPS23 enables selective translation of stress-responsive and virulence-associated mRNAs during host colonization and infection.

• Stress response integration: Similar to other ribosomal proteins, RPS23 likely functions beyond its structural role in ribosomes to modulate stress responses, particularly under the hypoxic conditions encountered during infection.

• Glutathione metabolism: Research indicates that glutathione metabolism is essential for C. glabrata virulence, and ribosomal proteins like RPS23 may influence this pathway through translational control of key enzymes .

• Biofilm formation: Preliminary evidence suggests that alterations in ribosomal protein expression, including RPS23, correlate with biofilm formation capacity, a key virulence trait of C. glabrata.

Given its essential nature, RPS23 itself is not typically categorized as a classical virulence factor, but rather as part of the core machinery that enables the expression of actual virulence determinants and stress adaptation proteins.

What role does RPS23 play in antifungal resistance mechanisms in Candida glabrata?

C. glabrata exhibits intrinsic resistance to several antifungals, with ribosomal proteins potentially contributing to this phenotype through several mechanisms:

Research strategies to investigate RPS23's specific role in resistance should include:

• CRISPR interference to modulate RPS23 expression without complete depletion

• Ribosome profiling in drug-sensitive vs. resistant strains

• Mass spectrometry to identify post-translational modifications in response to drug exposure

• Comparative analysis with closely related Candida species with different resistance profiles

These approaches could reveal whether RPS23 represents a potential target for adjuvant therapies to enhance antifungal efficacy.

How can protein-protein interaction networks involving RPS23 be characterized in C. glabrata?

Understanding the protein interaction network of RPS23 is crucial for elucidating its functions beyond the ribosome. Advanced methodologies for this characterization include:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Expressing tagged RPS23 in C. glabrata

  • Performing pull-downs under various growth conditions

  • Identifying co-purifying proteins by mass spectrometry

• Proximity labeling approaches:

  • Using BioID or APEX2 fusions with RPS23

  • Allowing in vivo labeling of proximal proteins

  • Purifying and identifying biotinylated proteins

• Three-dimensional structural predictions and hotspot analyses:
  Similar to methods used for SrbA interaction studies , computational prediction of protein structure and interaction interfaces can provide valuable insights:

  • Protein structure modeling using tools like AlphaFold

  • Interaction site prediction using tools similar to ClusPro and KFC2

  • Validation of predicted interactions by mutagenesis of key residues

• Crosslinking mass spectrometry (XL-MS):

  • Capturing transient interactions through chemical crosslinking

  • Identifying interaction sites at amino acid resolution

These approaches have revealed that transcription factors like SrbA can interact with various metabolic enzymes and proteins involved in cell cycle regulation , suggesting RPS23 might similarly engage in regulatory interactions beyond its structural role in ribosomes.

What are the most promising approaches for targeting RPS23 in antifungal development?

The essential nature and structural differences between fungal and human RPS23 make it a potential target for novel antifungal development. Promising approaches include:

• Structure-based drug design:

  • Solving high-resolution structures of C. glabrata RPS23

  • Identifying unique pockets or interfaces absent in human homologs

  • Virtual screening of compound libraries against these targets

  • Fragment-based approaches to develop highly specific inhibitors

• Peptide-based inhibitors:

  • Designing peptides that mimic natural binding partners

  • Targeting RPS23 interactions specific to fungal ribosomes

  • Employing stapled peptides for enhanced stability and cellular penetration

• RNA-targeted approaches:

  • Antisense oligonucleotides targeting RPS23 mRNA

  • Small molecules that disrupt RPS23 mRNA structure

• Combination strategies:

  • Identifying synergistic effects between RPS23 inhibitors and existing antifungals

  • Targeting multiple ribosomal proteins simultaneously

Screening methodologies should include both in vitro translation assays and whole-cell approaches to identify compounds with both target specificity and cellular efficacy.

What are the main technical challenges in studying C. glabrata RPS23 and how can they be overcome?

Researchers face several significant challenges when studying C. glabrata RPS23:

Emerging technologies to address these challenges include:

• Microfluidic approaches for single-cell analysis of ribosome function

• Nanobody development for specific detection and perturbation of RPS23 in its native context

• Time-resolved cryo-EM to capture dynamic states of ribosomes during translation

• In situ structural biology techniques to study RPS23 within intact cells

What future research directions will advance our understanding of RPS23 biology in pathogenic fungi?

Several promising research avenues could significantly advance our understanding of RPS23 biology:

• Specialized ribosomes: Investigating whether C. glabrata produces ribosomes with altered RPS23 states (modifications or paralogs) to selectively translate specific mRNAs during stress or host interaction.

• Moonlighting functions: Exploring potential extraribosomal roles of RPS23, particularly in signaling pathways relevant to pathogenesis, similar to how SrbA participates in multiple regulatory interactions .

• Host-pathogen interface: Examining whether RPS23 or ribosome-derived fragments interact with host factors during infection.

• Evolutionary adaptations: Comparative analysis across Candida species to identify pathogen-specific adaptations in RPS23 structure and function.

• Translational regulation networks: Mapping how RPS23 contributes to selective translation during stress response, potentially involving interaction with transcription factors like SrbA orthologs in C. glabrata .

Integration of multi-omics approaches—combining transcriptomics, proteomics, structural biology, and functional genetics—will be essential to fully elucidate the complex roles of RPS23 in fungal pathobiology.