Recombinant Candida glabrata U3 small nucleolar RNA-associated protein 25 (UTP25), partial

What is UTP25 and what is its role in Candida glabrata biology?

UTP25 (also known as Def) is a component of the SSU processome, a large ribonucleoprotein complex consisting of U3 snoRNA and at least 43 proteins. In Candida glabrata, UTP25 plays an essential role in pre-rRNA processing, specifically in the maturation of 18S rRNA.

Research has demonstrated that UTP25 is required for proper ribosome biogenesis. When UTP25 is depleted, cells exhibit accumulation of unprocessed 35S pre-rRNA precursor and a decrease in the abundance of 27SA pre-rRNA, indicating impaired pre-18S rRNA processing . This protein has been identified as essential for growth, as genetic depletion of UTP25 resulted in growth rate reduction comparable to depletion of other essential SSU processome components like Utp6 .

The importance of UTP25 extends beyond basic cellular functions, as it has been implicated in virulence-related processes and potentially in antifungal drug response mechanisms, making it a protein of significant interest in clinical and basic research settings.

How is UTP25 structurally and functionally characterized?

UTP25 has been classified as a "pseudohelicase" - a protein with structural similarities to DEAD-box RNA helicases but lacking functional helicase activity. Structural predictions using AlphaFold have revealed that human UTP25/DEF contains two structured globular domains (domain I: 157–559aa; domain II: 567–756aa) linked by a short hinge region (560–566aa) .

Despite displaying sequence similarity to DEAD-box RNA helicases, mutational studies have demonstrated that UTP25's DEAD-box motifs are dispensable for its function, confirming its classification as a pseudohelicase . This represents the first fully validated pseudohelicase, fulfilling all three criteria for classification as a pseudoenzyme: sequence similarity, lack of enzymatic function, and structural resemblance to authentic helicases.

As a pseudohelicase, UTP25 may function as a helicase co-factor, binding to pre-rRNA and recruiting an SSU processome helicase to specific sites. Evidence indicates that UTP25 forms direct protein-protein interactions with other known or suspected helicase co-factors such as Esf2, Lcp5, Pfa1, and Slx9, as well as with the helicase Dhr2 .

What is the broader significance of studying UTP25 in Candida glabrata?

Candida glabrata is the second most common cause of candidiasis globally, with clinical isolates often exhibiting high resistance to azole antifungals . This pathogen can thrive inside macrophages and demonstrates intrinsic tolerance to antifungal drugs, rendering infections a significant clinical challenge .

Understanding UTP25's function is valuable for several reasons:

• As an essential protein for growth, UTP25 provides insights into fundamental biological processes in fungi

• Its role in the SSU processome contributes to our understanding of ribosome biogenesis, a process critical for all living cells

• The discovery of UTP25 as a pseudohelicase enhances our knowledge of protein evolution and pseudoenzyme functions

• Studying UTP25 may reveal its potential role in virulence and/or drug resistance mechanisms

Research into UTP25 also contributes to the broader field of host-pathogen interactions, potentially leading to new therapeutic strategies against this increasingly important fungal pathogen.

What are the recommended methods for expressing and purifying recombinant Candida glabrata UTP25?

For successful expression and purification of recombinant C. glabrata UTP25, researchers should consider the following methodology:

Expression System Selection:

• E. coli BL21(DE3) strains are suitable for expression of full-length UTP25

• For expressing specific domains, consider using pET system vectors with N-terminal 6xHis-tags for easier purification

Optimized Expression Protocol:

• Transform expression plasmid into competent cells and plate on selective media

• Grow starter culture in LB medium with appropriate antibiotics at 37°C overnight

• Inoculate expression culture at 1:100 dilution and grow until OD600 reaches 0.6-0.8

• Induce protein expression with 0.5 mM IPTG and reduce temperature to 18°C for 16-18 hours

• Harvest cells by centrifugation (5,000 x g, 15 min, 4°C)

Purification Strategy:

• Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT with protease inhibitors)

• Lyse cells by sonication or high-pressure homogenization

• Clarify lysate by centrifugation (20,000 x g, 30 min, 4°C)

• Apply supernatant to Ni-NTA column equilibrated with lysis buffer

• Wash with buffer containing 20-40 mM imidazole

• Elute protein with buffer containing 250 mM imidazole

• Perform size exclusion chromatography for final purification

Special Considerations:

• UTP25 tends to migrate in glycerol gradients as a 165 kDa particle despite its actual 84 kDa size, suggesting it may form complexes or have an extended structure

• Include RNase inhibitors if studying RNA-binding properties

• For functional studies, verify protein integrity through circular dichroism or thermal shift assays

How can researchers effectively study UTP25's role in pre-rRNA processing?

To investigate UTP25's function in pre-rRNA processing, the following methodological approaches are recommended:

1. Conditional Depletion System:
Create a strain with UTP25 under control of a galactose-inducible/glucose-repressible promoter (GAL::3xHA-UTP25) to allow for controlled depletion of the protein. This approach was successfully used to demonstrate UTP25's essential role in pre-18S rRNA processing .

2. Northern Blot Analysis for Pre-rRNA Intermediates:

• Extract total RNA from cells at different time points after UTP25 depletion

• Separate RNA on formaldehyde-agarose gels

• Transfer to nylon membranes

• Hybridize with probes specific for different regions of the pre-rRNA

• Analyze accumulation of precursors and depletion of mature rRNAs

3. ChIP-Seq of RNA Polymerase II:
As demonstrated in research with C. glabrata during macrophage infection, ChIP-seq against elongating RNA Polymerase II can map transcriptional responses with high temporal resolution . This approach can be adapted to study how UTP25 deletion affects transcription of rRNA processing genes.

4. Co-Immunoprecipitation Analysis:
To identify proteins that interact with UTP25:

• Create tagged versions of UTP25 (e.g., HA-tagged)

• Perform immunoprecipitation using anti-tag antibodies

• Analyze co-precipitating proteins by mass spectrometry

• Validate interactions by reciprocal co-IPs

5. RNA-Protein Interaction Studies:
To study UTP25's interaction with pre-rRNA:

• Perform UV cross-linking immunoprecipitation (CLIP-seq)

• Analyze binding sites on pre-rRNA

• Correlate binding sites with pre-rRNA processing defects in UTP25-depleted cells

Example data from previous studies:
UTP25 depletion led to accumulation of 35S pre-rRNA and decrease in 27SA pre-rRNA, indicating a block in early pre-rRNA processing steps leading to 18S rRNA maturation .

What approaches can be used to investigate UTP25's potential role in antifungal resistance?

To explore whether UTP25 contributes to antifungal resistance in C. glabrata, researchers should consider a multi-faceted approach:

1. Gene Deletion and Complementation Studies:

• Generate UTP25 deletion mutants using homologous recombination

• Create complemented strains by reintroducing UTP25 on a plasmid

• Assess susceptibility to different classes of antifungals (azoles, echinocandins, polyenes) using standardized susceptibility testing methods

2. Transcriptomic Analysis:

• Compare gene expression profiles between wild-type and UTP25 mutant strains using RNA-seq

• Focus on known drug resistance genes (e.g., efflux pumps, ergosterol biosynthesis)

• Analyze data under normal conditions and upon exposure to sub-inhibitory concentrations of antifungals

3. Azole Accumulation Assays:
Similar to methods used in other C. glabrata studies, measure intracellular accumulation of fluorescent azole derivatives or radiolabeled antifungals in wild-type vs. UTP25 mutant cells .

4. Protein Interaction Studies:
Identify if UTP25 interacts with known regulators of drug resistance, such as:

• Transcription factors (e.g., CgPdr1)

• Efflux pumps (e.g., CgCdr1, CgCdr2)

• Components of stress response pathways

5. Evolution Experiments:

• Subject wild-type and UTP25 mutant strains to long-term exposure to increasing concentrations of antifungals

• Monitor development of resistance and identify genomic/transcriptomic changes

• Compare evolution trajectories between strains

This approach would be similar to the study on C. glabrata evolution from azole susceptibility to resistance through longstanding incubation with fluconazole .

How might UTP25's pseudohelicase nature contribute to its function in the SSU processome?

As a validated pseudohelicase, UTP25 presents an intriguing case for investigating how proteins that have lost their enzymatic function can gain alternative roles. Current research suggests several potential functional mechanisms:

Helicase Co-factor Hypothesis:
UTP25 may act as a regulatory co-factor for authentic helicases in the SSU processome. Evidence supporting this includes:

• Direct protein-protein interactions with the helicase Dhr2 and with known helicase co-factors Esf2, Lcp5, Pfa1, and Slx9

• Ability to bind to pre-rRNA, potentially recruiting helicases to specific sites

• Absence from stable SSU processome structures captured by cryoEM, consistent with the transient nature of helicase/co-factor interactions

Molecular Scaffold Hypothesis:
UTP25 may function as a structural scaffold in SSU processome assembly:

• Despite being an 84 kDa protein, UTP25 migrates in glycerol gradients as a 165 kDa particle, suggesting complex formation or extended structure

• This scaffolding role is consistent with known functions of other pseudoenzymes

• UTP25 could stabilize specific conformations of the pre-rRNA to facilitate processing

To test these hypotheses, researchers could:

• Perform structure-function analysis by generating UTP25 variants with mutations in potential RNA-binding or protein-interaction domains

• Use hydrogen-deuterium exchange mass spectrometry to identify conformational changes in UTP25 upon binding to RNA or protein partners

• Apply in vitro reconstitution approaches with purified UTP25 and authentic helicases to test for modulation of helicase activity

The study of UTP25 as a pseudohelicase has broader implications for understanding protein evolution through gene duplication and divergence, potentially informing predictions about the consequences of protein sequence variants in genetic diseases and cancer .

How does UTP25 interact with the RNA exosome and other RNA processing machinery?

The interaction between UTP25 and the RNA exosome represents an important area for investigation, especially considering recent findings about related proteins:

Current Knowledge and Research Gaps:

• Research on UTP3 has shown that it facilitates degradation of processed 5'ETS by recruiting the RNA exosome component EXOSC10 to the nucleolus

• Whether UTP25 has similar interactions with the RNA exosome remains unknown

• As a component of the SSU processome, UTP25 likely participates in a network of interactions with other RNA processing factors

Methodological Approaches for Investigation:

• Protein-Protein Interaction Mapping:

  • Implement BioID or proximity labeling approaches to identify proteins in close proximity to UTP25 in vivo

  • Perform yeast two-hybrid screens or pull-down assays to identify direct interacting partners

  • Use quantitative proteomics to compare interaction profiles in different conditions (e.g., normal growth vs. stress)

• Domain-Specific Interaction Analysis:
  Based on structural predictions that human UTP25/DEF contains two structured globular domains (domain I: 157–559aa; domain II: 567–756aa) , researchers could:

  • Generate domain-specific deletion or mutation constructs

  • Test each domain's interaction with putative partners

  • Identify which regions mediate specific interactions

• Subcellular Localization Studies:

  • Investigate whether UTP25 facilitates the nucleolar localization of other proteins, similar to UTP3's role

  • Use fluorescence microscopy with tagged proteins to track co-localization

  • Apply fluorescence recovery after photobleaching (FRAP) to analyze protein dynamics

• RNA-Protein Complexes Analysis:

  • Implement CLIP-seq or related techniques to identify RNA targets of UTP25

  • Compare with known targets of RNA exosome components

  • Identify potential overlap in binding sites that might indicate functional cooperation

Potential Experimental Design:
To test whether UTP25 interacts with the RNA exosome, researchers could:

• Create epitope-tagged versions of UTP25 and exosome components

• Perform co-immunoprecipitation experiments

• Analyze pre-rRNA processing defects in cells with mutations in both UTP25 and exosome components

• Look for genetic interactions (synthetic lethality or suppression) between UTP25 and exosome component mutations

How can researchers address the challenges of working with fungal proteins like UTP25 in heterologous expression systems?

Expressing and studying fungal proteins like UTP25 in heterologous systems presents several challenges that researchers must overcome:

Common Challenges and Solutions:

• Codon Usage Bias:

  • Challenge: Differences in codon usage between fungi and expression hosts (typically E. coli) can lead to poor expression.

  • Solution: Optimize codons for the expression host, particularly for rare codons. Alternatively, co-express rare tRNAs using specialized E. coli strains like Rosetta.

• Protein Solubility Issues:

  • Challenge: Fungal proteins often form inclusion bodies in bacterial systems.

  • Solutions:

    • Use fusion tags that enhance solubility (MBP, SUMO, GST)

    • Express at lower temperatures (16-18°C)

    • Co-express molecular chaperones

    • Consider eukaryotic expression systems (yeast, insect cells) for proteins resistant to bacterial expression

• Post-translational Modifications:

  • Challenge: Bacterial systems lack eukaryotic PTM machinery.

  • Solution: For proteins requiring PTMs, use eukaryotic expression systems like Pichia pastoris or insect cells.

• Protein Purification Challenges:

  • Challenge: UTP25 tends to migrate as a larger complex (165 kDa vs. actual 84 kDa) , suggesting it forms complexes or has an extended structure.

  • Solutions:

    • Include detergents or higher salt concentrations to disrupt non-specific interactions

    • Use stringent washing steps during affinity purification

    • Implement multi-step purification strategies, including ion exchange and size exclusion chromatography

Recommended Expression Protocol for UTP25:

• Generate constructs expressing different domains based on structural predictions:

  • Full-length UTP25

  • Domain I (approximately aa 157-559)

  • Domain II (approximately aa 567-756)

  • N-terminal region (aa 1-156)

• Test expression in multiple systems:

  • E. coli (BL21, Arctic Express, Rosetta)

  • Yeast (S. cerevisiae, P. pastoris)

  • Insect cells (if necessary)

• Optimize purification based on protein characteristics:

  • For studying RNA binding: Include RNase inhibitors and test for co-purifying RNAs

  • For structural studies: Include additional purification steps to ensure homogeneity

What are the best approaches for studying UTP25 in the context of fungal infection models?

Investigating UTP25's role in infection contexts requires specialized approaches that balance molecular detail with physiological relevance:

In Vitro Macrophage Infection Models:

Based on successful approaches used in C. glabrata research, the following protocol is recommended:

• Macrophage Preparation:

  • Culture THP-1 monocytes to 80% confluence

  • Resuspend cells to 10^6 cells/ml in complete RPMI medium

  • Add phorbol-13-myristyl-acetate (PMA) to 16 nM final concentration

  • Seed 1 million cells per well in 24-well plates

  • Incubate for 12 hours, replace medium, and allow 12-hour recovery

• Fungal Preparation:

  • Prepare wild-type C. glabrata and UTP25 conditional mutant strains

  • Adjust to 2×10^6 yeasts/ml in complete RPMI medium

  • Add 100 μL yeast suspension to each well of differentiated macrophages

  • After 2-hour co-incubation, wash three times with PBS to remove non-phagocytosed yeasts

• Analysis Methods:

  • At multiple time points (2h, 8h, 24h, 48h), lyse macrophages and plate dilutions to count fungal CFUs

  • Compare proliferation rates between wild-type and UTP25-depleted strains

  • Perform RNA extraction for transcriptomic analysis

  • Conduct microscopy to assess fungal morphology and localization within macrophages

In Vivo Galleria mellonella Model:

The G. mellonella (greater wax moth) larval model offers advantages for studying C. glabrata virulence:

• Infection Protocol:

  • Prepare C. glabrata cell suspensions of wild-type and UTP25 mutant strains

  • Inject approximately 5×10^7 CFU per larva

  • Monitor survival rates daily for 7-10 days

  • Recover hemolymph at different time points to assess fungal burden

• Data Analysis:

  • Generate Kaplan-Meier survival curves

  • Compare fungal proliferation in hemolymph across strains

  • Assess statistical significance using appropriate tests (log-rank test for survival, t-test or ANOVA for CFU counts)

Molecular Analysis During Infection:

To understand UTP25's function during infection:

• Design a system to isolate C. glabrata RNA from infected hosts with minimal host contamination

• Implement ChIP-seq against elongating RNA Polymerase II to map transcriptional responses with high temporal resolution

• Compare results between wild-type and UTP25-depleted strains to identify UTP25-dependent processes

How can researchers interpret conflicting data regarding UTP25's function across different experimental systems?

When studying complex proteins like UTP25, researchers often encounter seemingly contradictory results. Here's a methodological framework for addressing and interpreting such conflicts:

Sources of Experimental Variation:

• Genetic Background Differences:

  • C. glabrata shows substantial genetic diversity, with at least 20 separate sequence types identified across global isolates

  • Even within the same species, reference strains can have significant differences (e.g., CBS138 vs strain 2001)

• Methodological Variations:

  • Different detection methods have varied sensitivity and specificity

  • Tagging strategies may affect protein function differently

  • Growth conditions can significantly influence results

• Temporal Considerations:

  • C. glabrata mounts chronological transcriptional responses during infection

  • Short-term vs. long-term experiments may yield different results

Systematic Approach to Resolving Conflicts:

• Perform Control Experiments:

  • Test multiple genetic backgrounds

  • Use different tagging strategies and confirm functionality

  • Validate key results using independent methodologies

• Implement Rigorous Statistical Analysis:

  • Apply appropriate statistical tests

  • Consider multiple hypothesis testing correction

  • Calculate effect sizes, not just statistical significance

• Utilize Meta-Analysis Techniques:

  • Aggregate data across studies

  • Weight results based on sample size and methodological rigor

  • Identify patterns that persist across experimental variations

Decision Framework for Conflicting Results:

Statistical Considerations:
When interpreting possibly conflicting results, researchers should implement rigorous statistical frameworks:

• Control for false discovery rate using methods like Bonferroni correction (e.g., adjusting alpha to 0.001 for multiple comparisons)

• Consider power analysis to ensure sample sizes are sufficient to detect effects of interest

• Report effect sizes alongside p-values to better characterize the magnitude of differences

What are the most promising avenues for future research on UTP25 and its role in Candida glabrata biology?

Based on current knowledge gaps and potential significance, the following research directions appear most promising:

1. Structure-Function Relationships:

• Obtain high-resolution structural data for UTP25 through X-ray crystallography or cryo-EM

• Compare with authentic RNA helicases to identify structural features unique to pseudohelicases

• Map RNA binding sites and protein interaction domains

• Use this information to design specific inhibitors as potential antifungal targets

2. Evolutionary Analysis:

• Perform comparative analysis of UTP25 across fungal species, especially pathogenic vs. non-pathogenic species

• Investigate whether UTP25 shows signatures of positive selection in clinical isolates

• Examine the evolution of pseudohelicase function from ancestral helicases

3. Systems Biology Approaches:

• Integrate transcriptomic, proteomic, and functional data to position UTP25 within cellular networks

• Identify condition-specific roles during stress response and host interaction

• Apply network analysis to predict additional functions and interactions

4. Translational Research:

• Assess whether UTP25 could serve as a biomarker for specific C. glabrata infections

• Investigate UTP25 as a potential drug target, exploiting its essential nature

• Develop screening assays for compounds that disrupt UTP25 function or interactions

5. Host-Pathogen Interactions:

• Study how host immune responses affect UTP25 expression and function

• Investigate whether UTP25 contributes to immune evasion mechanisms

• Examine UTP25's role in adaptation to different host niches

How might knowledge about UTP25 contribute to developing new antifungal strategies?

The emergence of drug-resistant Candida strains necessitates novel antifungal approaches. UTP25 offers several promising avenues for therapeutic development:

Target Validation Considerations:

• Essential Nature:

  • UTP25 is essential for growth, making it an attractive drug target

  • Genetic depletion of UTP25 causes significant growth defects

  • As a processome component, it likely has no direct mammalian homolog with identical function

• Evolutionary Distinctiveness:

  • As a pseudohelicase, UTP25 likely has unique structural features

  • These distinct features could allow for selective targeting without affecting human proteins

• Potential Role in Virulence and Resistance:

  • If UTP25 contributes to stress response or drug resistance mechanisms, targeting it might enhance existing antifungals

  • Combination therapy approaches could be particularly effective

Potential Therapeutic Strategies:

• Small Molecule Inhibitors:

  • Target UTP25 protein-protein interactions, especially with known partners like Dhr2 helicase

  • Disrupt UTP25-RNA interactions essential for processome function

  • Design allosteric inhibitors that lock UTP25 in inactive conformations

• Peptide-Based Therapeutics:

  • Develop peptides that mimic natural binding partners but disrupt function

  • Create cell-penetrating peptides that interfere with UTP25 localization

• Immunotherapeutic Approaches:

  • If UTP25 has cell surface exposure during infection, develop antibodies or immunomodulators

  • Target UTP25-dependent processes specifically activated during infection

• Combination Therapies:

  • Identify synergistic interactions between UTP25 inhibition and existing antifungals

  • Similar to approaches used with sphingosine biosynthesis inhibitors (e.g., SDZ 90-215 or myriocin) that enhanced micafungin potency in C. glabrata

Development Pathway:

• High-throughput screening for compounds that bind UTP25

• Secondary screens for inhibition of UTP25 function in cell-free systems

• Tertiary screens in C. glabrata to confirm antifungal activity

• Optimization for selectivity, potency, and pharmacological properties

• In vivo testing in infection models

What novel experimental techniques could advance our understanding of UTP25 function?

Emerging technologies offer new opportunities to explore UTP25 biology at unprecedented resolution:

1. Advanced Structural Biology Approaches:

• Cryo-Electron Tomography: Visualize UTP25 within the native cellular environment

• Integrative Structural Biology: Combine X-ray crystallography, NMR, and computational modeling

• AlphaFold-Enabled Structure Prediction: Leverage recent advances in protein structure prediction to guide experimental design

2. Single-Cell and Spatial Technologies:

• Single-Cell RNA-Seq: Profile transcriptional heterogeneity in UTP25 response during infection

• Spatial Transcriptomics: Map UTP25-dependent gene expression in the context of host tissues

• Super-Resolution Microscopy: Track UTP25 localization and dynamics at nanometer resolution

3. CRISPR-Based Technologies:

• CRISPRi/CRISPRa: Create tunable repression or activation of UTP25 and interacting genes

• Base Editing: Introduce precise mutations to test structure-function hypotheses

• CRISPR Screens: Identify genetic interactions through genome-wide screens in UTP25 mutant backgrounds

4. Protein Engineering Approaches:

• Optogenetic Control: Create light-activatable UTP25 variants to control function with temporal precision

• Chemical Genetics: Develop rapidly inducible degradation systems for acute UTP25 depletion

• Proximity Labeling: Map the UTP25 interactome in different cellular compartments and conditions

5. High-Resolution 'Omics Integration:

• Multi-omics Analysis: Integrate transcriptomics, proteomics, and metabolomics data from UTP25 mutants

• Ribosome Profiling: Examine translation effects of UTP25 perturbation with codon-level resolution

• Epitranscriptomics: Investigate how UTP25 affects RNA modifications important for ribosome assembly

Implementation Strategy:
When applying these novel techniques, researchers should consider:

• Starting with well-controlled model systems before moving to complex infection scenarios

• Validating findings using complementary approaches

• Developing computational pipelines to integrate multi-modal data

• Establishing collaborations that bring together expertise across disciplines