Recombinant Candida glabrata Ribosome biogenesis protein ERB1 (ERB1), partial

How does ERB1 protein structure relate to its function in ribosome assembly?

ERB1 in C. glabrata contains characteristic WD40 repeat domains that form a β-propeller structure, enabling multiple protein-protein interactions crucial for ribosome assembly. These domains facilitate ERB1's role as a scaffold protein during pre-ribosome formation, allowing it to interact with various assembly factors and ribosomal proteins simultaneously. The functional domains of ERB1 are highly conserved across fungal species, though species-specific variations exist that may contribute to differences in ribosome assembly pathways between C. glabrata and other yeast species like S. cerevisiae . These structural characteristics enable ERB1 to participate in the complex, hierarchical assembly process of the pre-60S ribosomal subunit.

What experimental systems are available for studying ERB1 function in C. glabrata?

Several experimental approaches can be employed to study ERB1 function in C. glabrata:

• Genetic Manipulation Systems: CRISPR-Cas9 ribonucleoprotein (RNP)-based systems coupled with drug resistance cassettes (NatMX/SAT1, HphMX, KanMX, or BleMX) allow for targeted gene deletions, epitope tagging, and rescue construct implementation in C. glabrata .

• Recombinant Protein Expression: Baculovirus expression systems can be used to produce recombinant ERB1 protein with high purity (>85% by SDS-PAGE) for biochemical and structural studies .

• Functional Complementation Assays: Replacement of ERB1 with orthologous genes from related species like S. cerevisiae or C. albicans can reveal species-specific functions and evolutionary adaptations .

• Ribosome Profiling: This technique can be used to analyze the impact of ERB1 mutations on ribosome assembly and function by examining ribosome distribution on mRNAs.

How can researchers optimize the reconstitution of recombinant ERB1 protein for functional studies?

For optimal reconstitution of recombinant C. glabrata ERB1 protein:

• Initial Preparation: Briefly centrifuge the protein vial before opening to bring contents to the bottom.

• Reconstitution Protocol:

  • Dissolve lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is recommended as default)

  • Aliquot for long-term storage at -20°C/-80°C

• Storage Considerations:

  • Liquid form typically maintains stability for 6 months at -20°C/-80°C

  • Lyophilized form typically maintains stability for 12 months at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Quality Control: Verify protein integrity via SDS-PAGE before experimental use, ensuring purity remains >85%.

These methodological considerations are critical for maintaining ERB1 protein functionality, as improper reconstitution or storage can compromise protein activity and experimental results.

What approaches can be used to study interactions between ERB1 and other ribosome assembly factors in C. glabrata?

Several methodological approaches can be implemented to elucidate interactions between ERB1 and other assembly factors:

• Co-immunoprecipitation (Co-IP): Using epitope-tagged ERB1 (enabled by CRISPR-based genetic tools for C. glabrata), researchers can identify protein interaction partners through pull-down assays followed by mass spectrometry .

• Yeast Two-Hybrid (Y2H) Screening: This can reveal direct protein-protein interactions between ERB1 and candidate ribosome assembly factors.

• Proximity-based Labeling: BioID or APEX2 fusions to ERB1 can identify neighboring proteins in the native cellular environment.

• Cryo-electron Microscopy: This can visualize ERB1 within the context of pre-ribosomal particles, providing structural insights into its interactions with rRNA and other assembly factors.

• Genetic Interaction Studies: Synthetic genetic array analysis using the expanded toolkit of drug resistance cassettes can identify functionally related genes .

Each approach provides complementary information about the molecular network surrounding ERB1 during ribosome biogenesis in C. glabrata.

How can researchers design experiments to investigate species-specific functions of ERB1 between C. glabrata and related Candida species?

Experimental design for comparative studies of ERB1 function across Candida species should incorporate these methodological approaches:

• Chimeric Protein Construction: Create fusion proteins with domains from ERB1 orthologs across species (C. glabrata, C. albicans) to map functional regions responsible for species-specific activities.

• Heterologous Complementation: Express C. glabrata ERB1 in C. albicans (and vice versa) to assess functional conservation and divergence. This approach revealed that replacement of certain proteins with orthologs from different species can enhance incorporation of assembly factors into pre-ribosomes .

• Evolutionary Rate Analysis: Compare sequence conservation rates of ERB1 across fungal species to identify rapidly evolving regions that may contribute to species-specific functions.

• Ribosome Assembly Assays: Compare pre-rRNA processing patterns and pre-ribosome composition between species to identify differences in the ribosome assembly pathway that might be influenced by ERB1.

• Comparative Structural Studies: Use computational modeling and experimental structure determination to identify structural differences in ERB1 that might explain functional divergence.

This multi-faceted approach can reveal how ERB1 has evolved to accommodate species-specific variations in ribosome structure and assembly pathways.

How does C. glabrata ERB1 contribute to species-specific variations in ribosome biogenesis compared to S. cerevisiae?

Candida glabrata ERB1 contributes to species-specific ribosome biogenesis in several key ways:

• rRNA Expansion Segment Interaction: ERB1 in C. glabrata has evolved to interact with species-specific rRNA expansion segments (ESs), which differ between C. glabrata and S. cerevisiae. These interactions are critical for proper pre-ribosome assembly and maturation .

• Assembly Factor Recruitment: C. glabrata ERB1 recruits species-specific assembly factors that may differ from those in S. cerevisiae, contributing to differences in the ribosome assembly pathway between the two species.

• Co-evolutionary Adaptation: Research suggests that assembly factors like ERB1 co-evolved with species-specific rRNA variations, specializing the ribosome biogenesis pathway across different yeast species . This co-evolution ensures proper ribosome synthesis despite differences in rRNA structure.

• Functional Conservation despite Sequence Divergence: Despite sequence differences, ERB1 maintains its core function in ribosome biogenesis across species, highlighting the protein's adaptability to species-specific ribosomal contexts.

Understanding these species-specific functions of ERB1 provides insights into the evolution of ribosome biogenesis pathways and how pathogens like C. glabrata have adapted these essential processes.

What methodologies are most effective for studying the role of ERB1 in pre-rRNA processing in C. glabrata?

For investigating ERB1's role in pre-rRNA processing, researchers should consider these methodological approaches:

• Northern Blot Analysis: This technique allows visualization of specific pre-rRNA intermediates using probes targeting various regions of the rRNA precursor. Changes in the pattern of pre-rRNA species in ERB1 mutants can reveal the step at which processing is blocked.

• Pulse-Chase Labeling: By pulse-labeling newly synthesized RNA with radioactive precursors followed by a chase period, researchers can track the kinetics of rRNA processing and maturation in wild-type versus ERB1-mutant strains.

• Primer Extension Analysis: This method can map precise cleavage sites in pre-rRNA and identify processing defects caused by ERB1 mutations.

• RNA-Sequencing: High-throughput sequencing of rRNA species can provide a comprehensive view of processing intermediates and their abundance in different genetic backgrounds.

• Sucrose Gradient Analysis: This technique separates pre-ribosomal particles based on size, allowing researchers to determine which pre-ribosomal complexes are affected by ERB1 dysfunction.

• Conditional Depletion Systems: Creating conditional ERB1 mutants (using inducible promoters or degron tags) enables time-course studies of pre-rRNA processing after ERB1 depletion.

Each of these approaches provides complementary information about ERB1's specific role in the complex pre-rRNA processing pathway.

How can researchers investigate the potential connection between ribosome biogenesis defects and antifungal resistance in C. glabrata?

Investigating links between ribosome biogenesis and antifungal resistance requires integrated approaches:

• Gene Expression Analysis: Compare expression levels of ERB1 and other ribosome biogenesis factors in azole-resistant versus susceptible C. glabrata strains. Recent research has shown that transcription factors like Hap1A and Hap1B, which are involved in ergosterol biosynthesis and azole resistance, may indirectly affect ribosome biogenesis .

• Genetic Interaction Studies: Utilize CRISPR-based tools with multiple drug resistance markers (NatMX, HphMX, KanMX, BleMX) to create double mutants lacking both ERB1 and genes involved in antifungal resistance pathways, such as ERG3 or ERG5 .

• Ribosome Profiling of Resistant Strains: Apply ribosome footprinting to examine translational changes in resistant strains, particularly focusing on genes involved in ergosterol biosynthesis and drug efflux.

• Targeted Drug Sensitivity Assays: Test ERB1 mutant strains for altered sensitivity to different classes of antifungals, including azoles, echinocandins, and polyenes.

• Metabolic Labeling Studies: Investigate whether ERB1 mutations affect translation of specific mRNAs involved in drug resistance mechanisms, such as ergosterol biosynthesis genes or drug transporters.

These approaches can reveal whether alterations in ribosome biogenesis, potentially through ERB1 dysfunction, contribute to the complex phenotype of antifungal resistance in C. glabrata.

What are the key considerations for designing CRISPR-Cas9 targeting strategies for ERB1 manipulation in C. glabrata?

When designing CRISPR-Cas9 strategies for ERB1 manipulation in C. glabrata, researchers should consider:

• Guide RNA Selection:

  • Target unique sequences in ERB1 to avoid off-target effects

  • Use algorithms to predict guide RNA efficiency and specificity

  • Consider the chromosomal context of ERB1 to ensure accessibility

• Delivery Method:

  • In vitro assembled ribonucleoprotein (RNP) complexes show high efficiency in C. glabrata

  • Optimize transformation protocols for C. glabrata, which may differ from those used for S. cerevisiae

• Selection Strategy:

  • Utilize the expanded toolkit of drug resistance cassettes (NatMX/SAT1, HphMX, KanMX, BleMX) for marker selection

  • Design cassettes with homology arms of appropriate length (40-60 bp) for efficient homologous recombination

• Verification Methods:

  • PCR-based genotyping to confirm successful editing

  • Sequencing to verify the precise modification

  • Western blotting to confirm protein alteration (for epitope tagging)

• Functional Validation:

  • Include rescue experiments with wild-type ERB1 to confirm phenotype specificity

  • Consider complementation with orthologous genes from related species to identify species-specific functions

These considerations ensure efficient and specific genetic manipulation of ERB1 in C. glabrata for functional studies.

What are the most reliable methods for quantifying ribosome biogenesis defects in ERB1 mutant strains?

For reliable quantification of ribosome biogenesis defects in ERB1 mutants:

• Polysome Profiling:

  • Analyze cytoplasmic extracts on sucrose gradients to quantify free 40S, 60S, 80S ribosomes, and polysomes

  • Calculate 60S/40S ratios as indicators of large subunit biogenesis defects

  • Measure polysome/monosome ratios to assess global translation efficiency

• rRNA Processing Analysis:

  • Quantitative Northern blotting with phosphorimager analysis

  • qRT-PCR to measure specific pre-rRNA species

  • High-throughput RNA sequencing for comprehensive rRNA intermediate profiling

• Ribosomal Protein Incorporation:

  • Western blotting to assess levels of specific ribosomal proteins in mature ribosomes

  • Mass spectrometry-based proteomics to analyze ribosome composition

  • Fluorescence microscopy of GFP-tagged ribosomal proteins to track localization

• Growth Rate Measurements:

  • Automated growth curve analysis under various conditions

  • Colony size measurements on solid media

  • Competition assays to detect subtle growth defects

• Translation Fidelity Assays:

  • Reporter systems to measure stop codon readthrough, frameshifting, and misincorporation rates

  • In vitro translation assays using purified ribosomes from mutant strains

These approaches provide complementary quantitative measures of ribosome biogenesis defects at different levels, from rRNA processing to functional translation.

How can researchers optimize storage and handling conditions for recombinant ERB1 protein to maintain its structural integrity?

Optimal storage and handling of recombinant ERB1 protein requires attention to several key parameters:

• Storage Temperature:

  • Store at -20°C/-80°C for long-term stability

  • Liquid formulations maintain stability for approximately 6 months

  • Lyophilized formulations maintain stability for approximately 12 months

• Buffer Composition:

  • Include glycerol (5-50%) as a cryoprotectant, with 50% being the recommended default concentration

  • Consider adding protease inhibitors to prevent degradation

  • Optimize pH and salt concentration based on ERB1's isoelectric point

• Aliquoting Strategy:

  • Prepare single-use aliquots to avoid repeated freeze-thaw cycles

  • Working stocks can be maintained at 4°C for up to one week

  • Document the number of freeze-thaw cycles for each aliquot

• Handling Precautions:

  • Briefly centrifuge vials before opening to collect contents at the bottom

  • Avoid vortexing, which can cause protein denaturation

  • Use low-protein-binding tubes and pipette tips

• Quality Control Measures:

  • Regularly verify protein integrity by SDS-PAGE

  • Monitor activity using functional assays specific to ERB1

  • Check for aggregation using dynamic light scattering or size-exclusion chromatography

Adhering to these guidelines ensures that experimental outcomes reflect the true biological properties of ERB1 rather than artifacts caused by protein degradation or denaturation.

How can researchers integrate bioinformatic and experimental approaches to study the evolution of ERB1 function across Candida species?

An integrated approach to studying ERB1 evolution across Candida species should combine:

• Phylogenetic Analysis:

  • Construct phylogenetic trees of ERB1 sequences from various Candida species

  • Identify conserved domains and species-specific variations

  • Calculate evolutionary rates for different protein regions

• Structural Bioinformatics:

  • Generate homology models of ERB1 from different species

  • Map conserved and variable regions onto 3D structures

  • Predict functional consequences of species-specific amino acid substitutions

• Experimental Validation:

  • Create chimeric proteins with domains from different species

  • Test functionality of these chimeras in multiple Candida species

  • Use heterologous complementation to determine functional conservation

• Comparative Genomics:

  • Analyze synteny and gene neighborhood of ERB1 across species

  • Identify co-evolved genes that may functionally interact with ERB1

  • Examine selection pressures on ERB1 and interacting partners

• Transcriptomics Integration:

  • Compare expression patterns of ERB1 and related genes across species

  • Identify species-specific regulatory mechanisms

  • Correlate expression with phenotypic differences in ribosome biogenesis

This integrative approach reveals how evolutionary processes have shaped ERB1 function and contributes to our understanding of species-specific adaptations in ribosome biogenesis pathways.

What experimental approaches can reveal the potential role of ERB1 in stress response and adaptation in C. glabrata?

To investigate ERB1's role in stress response:

• Stress Condition Profiling:

  • Expose wild-type and ERB1 mutant C. glabrata to various stressors (oxidative, osmotic, pH, antifungal drugs)

  • Measure growth rates, viability, and morphological changes

  • Compare stress-induced transcriptional responses using RNA-seq

• Host-Relevant Conditions:

  • Test growth under iron limitation, nutrient restriction, and hypoxic conditions that mimic host environments

  • Examine ERB1 mutant behavior in macrophage co-culture systems

  • Assess adaptation to specific host niches using ex vivo infection models

• Ribosome Heterogeneity Analysis:

  • Investigate whether stress conditions alter ERB1 function or localization

  • Examine if specialized ribosomes are produced under stress through ERB1-dependent mechanisms

  • Analyze translational reprogramming during stress response in ERB1 mutants

• Genetic Interaction Mapping:

  • Screen for synthetic interactions between ERB1 and stress response genes

  • Utilize the expanded toolkit of drug resistance cassettes for genetic manipulation

  • Create double mutants lacking both ERB1 and stress response factors

• Post-Translational Modification Analysis:

  • Examine whether ERB1 undergoes stress-induced modifications

  • Map modification sites and their impact on protein function

  • Identify enzymes responsible for these modifications

These approaches can reveal whether ERB1 contributes to stress adaptation beyond its canonical role in ribosome biogenesis, potentially connecting ribosome function to environmental adaptation.

How might understanding ERB1 function contribute to novel antifungal strategies against C. glabrata infections?

ERB1's potential as an antifungal target offers several research directions:

• Target Validation Approaches:

  • Create conditional ERB1 mutants to confirm essentiality under infection-relevant conditions

  • Assess virulence of ERB1-depleted strains in animal models

  • Identify minimum inhibitory levels of ERB1 activity required for viability

• Species-Specific Targeting:

  • Compare structural differences between fungal and human ERB1 orthologs

  • Identify functionally divergent regions that could be selectively targeted

  • Design assays to screen for compounds that exploit these differences

• Combination Therapy Exploration:

  • Test whether ERB1 inhibition sensitizes C. glabrata to existing antifungals

  • Investigate synergy with azoles, given C. glabrata's intrinsic resistance mechanisms

  • Explore connections to ergosterol biosynthesis, as recent research shows links between ribosome biogenesis and azole resistance pathways

• Drug Screening Strategies:

  • Develop high-throughput assays based on ERB1 function or protein-protein interactions

  • Screen chemical libraries for compounds that disrupt ERB1 activity

  • Utilize in silico approaches to identify potential binding pockets

• Resistance Mechanism Analysis:

  • Characterize potential resistance mechanisms to ERB1-targeting compounds

  • Determine the frequency of resistance emergence

  • Identify genetic alterations that confer resistance to ERB1 inhibition

Research in these areas could identify novel vulnerabilities in C. glabrata, potentially addressing the challenge of intrinsic and acquired antifungal resistance in this important human pathogen.