Recombinant Candida glabrata Nucleolar protein 9 (NOP9), partial

What is Nucleolar Protein 9 (NOP9) in Candida glabrata and what is its function?

NOP9 in C. glabrata is an essential nucleolar protein involved in ribosome biogenesis, specifically in the maturation of pre-18S rRNA. It functions as a critical RNA-binding protein present in both 90S and 40S pre-ribosomes . NOP9 contains multiple pumilio-like RNA binding repeats that enable it to recognize specific sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease Nob1 .

The primary function of NOP9 is to regulate the timing of pre-rRNA processing by preventing premature cleavage. When NOP9 is depleted in cells, early cleavages of the 35S pre-rRNA are inhibited, resulting in nucleolar retention of accumulated precursors and failure to synthesize 18S rRNA . This positions NOP9 as an essential factor in nuclear maturation of 90S and pre-40S ribosomal subunits.

How is the structure of C. glabrata NOP9 characterized?

The structural characterization of NOP9 reveals:

• A distinct 'C'-shaped fold formed from 11 Pumilio repeats

• Crystal structure determined at 2.1-Å resolution

• Multiple pumilio-like putative RNA binding domains that display robust in vitro RNA binding activity

• Structural features that allow it to recognize both sequence and structural elements in the 20S pre-rRNA

This structural arrangement facilitates NOP9's role in binding to pre-rRNA and preventing premature cleavage by the nuclease Nob1, thereby ensuring correct processing of pre-18S rRNA .

Why is C. glabrata NOP9 important in pathogenicity research?

C. glabrata is an opportunistic human fungal pathogen accounting for up to 29% of total Candida bloodstream infections . As an essential protein involved in ribosome biogenesis, NOP9 represents:

• A potential target for antifungal development, especially given C. glabrata's increasing resistance to common antifungals

• A model for understanding fundamental cellular processes that may contribute to virulence

• An opportunity to explore species-specific ribosome biogenesis in this important pathogen

Research on C. glabrata is particularly relevant for elderly individuals, diabetic patients, and organ transplant recipients who are at increased risk for C. glabrata infections .

What are the standard methodologies for expressing recombinant C. glabrata NOP9?

Expression of recombinant C. glabrata NOP9 typically involves:

Expression Systems:

• Heterologous expression in E. coli for structural studies and biochemical assays

• Homologous expression in C. glabrata or S. cerevisiae for functional studies

Expression Strategy:

• PCR amplification of the NOP9 coding sequence from C. glabrata genomic DNA

• Cloning into appropriate expression vectors containing:

  • Inducible promoters (e.g., copper-inducible MTI promoter for C. glabrata)

  • Fusion tags for purification (His, GST, or MBP)

• Transformation into expression host

• Induction of protein expression

• Purification via affinity chromatography

Experimental Example:
For homologous expression in C. glabrata, researchers have successfully utilized the copper-inducible MTI promoter system, as demonstrated in studies of other C. glabrata proteins . The MTI promoter can be generated by PCR with specific primers containing regions homologous to the target vector flanking regions, facilitating recombination-based cloning .

How can researchers verify the RNA-binding activity of recombinant NOP9?

RNA-binding activity of recombinant NOP9 can be assessed through several complementary approaches:

In vitro RNA binding assays:

• Electrophoretic mobility shift assays (EMSA): Mixing purified recombinant NOP9 with labeled RNA fragments containing putative binding sites from 20S pre-rRNA, followed by native gel electrophoresis to detect mobility shifts indicating protein-RNA complex formation.

• Filter binding assays: Quantitative measurement of RNA binding by passing protein-RNA mixtures through nitrocellulose filters, which retain protein-bound RNA.

• Surface plasmon resonance (SPR): Real-time measurement of binding kinetics between immobilized NOP9 and flowing RNA ligands.

RNA-protein crosslinking:

• UV crosslinking of NOP9-RNA complexes followed by immunoprecipitation and RNA sequencing to identify binding sites in vivo.

Structural analysis:

• Small-angle X-ray scattering (SAXS) to generate models of NOP9:RNA complexes, as demonstrated in previous studies .

Functional verification:

• Complementation assays in NOP9-depleted cells to determine if RNA-binding mutations affect pre-rRNA processing.

What experimental systems are used to study C. glabrata NOP9 function in vivo?

Several experimental systems can be employed to study C. glabrata NOP9 function:

Genetic manipulation in C. glabrata:

• Targeted gene deletion using homologous recombination approaches

• Conditional expression systems using regulated promoters

• CRISPR-Cas9 mediated genome editing for precise mutations

Expression analysis:

• RT-PCR and quantitative PCR to measure NOP9 expression levels

• Northern blotting to analyze pre-rRNA processing defects

Infection models:

• Galleria mellonella larval infection model, which has been successfully used for C. glabrata virulence studies

• Murine models of systemic candidiasis or organ-specific infections

• Cell culture infection models using macrophages (e.g., THP1 cell line) to assess intracellular replication

Pre-ribosome analysis:

• Sucrose gradient centrifugation to isolate pre-ribosomal particles

• Mass spectrometry analysis of NOP9-associated proteins

• RNA-seq to characterize pre-rRNA processing defects

How does C. glabrata NOP9 compare structurally and functionally to NOP9 in other fungal species?

Comparative analysis reveals both conservation and divergence in NOP9 structure and function:

The conservation of NOP9 across fungal species highlights its fundamental role in ribosome biogenesis, while species-specific differences may contribute to unique aspects of rRNA processing in each organism. These differences could potentially be exploited for species-specific antifungal development .

What are the technical challenges in expressing and purifying functional recombinant C. glabrata NOP9?

Researchers face several challenges when working with recombinant C. glabrata NOP9:

Solubility issues:

• NOP9's multiple RNA-binding domains can lead to aggregation or inclusion body formation

• Solution: Optimization of expression conditions (temperature, induction time), use of solubility-enhancing fusion tags (MBP, SUMO), or refolding protocols

Maintaining RNA-binding activity:

• Purification processes may disrupt the native conformation needed for RNA binding

• Solution: Inclusion of RNA competitors during purification, reducing salt concentration in buffers, or co-expression with interaction partners

Post-translational modifications:

• Fungal-specific modifications may be absent in bacterial expression systems

• Solution: Expression in eukaryotic systems like yeast or insect cells

Structural integrity:

• The complex 'C'-shaped fold may be difficult to maintain during purification

• Solution: Stabilizing buffer components, careful optimization of purification conditions

Experimental design considerations:

• Include functional assays at each purification step to track activity

• Develop robust storage conditions to prevent activity loss

• Consider expressing functional domains separately if full-length protein proves problematic

How does NOP9 integrate into the broader pre-rRNA processing machinery in C. glabrata?

NOP9 functions within a complex network of pre-rRNA processing factors:

Temporal regulation:

• NOP9 prevents premature cleavage by the nuclease Nob1

• This timing control ensures proper sequential processing of pre-rRNA

Spatial organization:

• NOP9 is present in both 90S and 40S pre-ribosomes

• This suggests a role spanning early to late stages of small subunit maturation

Protein-protein interactions:

• NOP9 likely interacts with other small subunit processing factors

• These interactions coordinate processing events and structural rearrangements

RNA recognition:

• NOP9 binds specific RNA motifs near Nob1 cleavage sites

• This binding may induce structural changes that regulate accessibility to processing enzymes

Evolutionary conservation:

Understanding these integration points is crucial for developing a comprehensive model of ribosome biogenesis in C. glabrata and identifying potential intervention points for antifungal development.

What evidence exists for NOP9's role in C. glabrata virulence and pathogenicity?

While direct evidence for NOP9's role in C. glabrata virulence remains limited, several lines of reasoning suggest potential connections:

Essential cellular function:

• As a key factor in ribosome biogenesis, NOP9 is essential for cellular growth and proliferation

• Growth rate and protein synthesis capacity directly impact virulence potential

Comparison to other ribosome biogenesis factors:

• Several ribosomal proteins and processing factors have been linked to virulence in fungal pathogens

• For example, studies in C. glabrata have shown that some ribosome-associated proteins like CgDtr1 affect virulence in the G. mellonella infection model

Stress adaptation:

• Ribosome biogenesis is regulated during stress responses

• C. glabrata encounters various stresses in the host, including oxidative stress within macrophages

• Proper regulation of pre-rRNA processing may be crucial for stress adaptation

Host-pathogen interface:

• C. glabrata must adapt to the host environment, which may require modulation of ribosome biogenesis

• NOP9's role in controlling 18S rRNA synthesis could influence adaptation to different host niches

Future studies using conditional NOP9 mutants in virulence models would help establish direct connections between NOP9 function and pathogenicity.

How might structural analysis of C. glabrata NOP9 inform antifungal drug development?

Structural characterization of C. glabrata NOP9 offers several promising avenues for antifungal development:

Structure-based drug design:

• The 2.1-Å crystal structure provides a foundation for in silico screening of small molecule inhibitors

• The 'C'-shaped fold with multiple Pumilio repeats offers potential binding pockets for small molecules

RNA-protein interface targeting:

• Understanding how NOP9 recognizes its RNA targets could lead to competitive inhibitors

• Small molecules or peptides that mimic RNA structure could disrupt this essential interaction

Species selectivity:

• Comparing NOP9 structures across fungal and human homologs could identify fungal-specific features

• These differences could be exploited to develop selective inhibitors with minimal host toxicity

Resistance considerations:

• C. glabrata shows increasing resistance to standard antifungals like azoles

• Novel targets like NOP9 could bypass existing resistance mechanisms

• Structure-function studies could predict potential resistance mutations

Combination approaches:

• Understanding how NOP9 inhibition affects cellular physiology could inform combination therapies

• Targeting multiple steps in ribosome biogenesis might produce synergistic effects

What methodological approaches can be used to investigate NOP9's interactions with other ribosome biogenesis factors?

Several complementary approaches can elucidate NOP9's interaction network:

Biochemical approaches:

• Co-immunoprecipitation (Co-IP): Using tagged NOP9 to pull down interaction partners, followed by mass spectrometry identification

• Yeast two-hybrid screening: Systematic identification of protein-protein interactions

• Proximity labeling: BioID or APEX2 fusion proteins to identify proteins in close proximity to NOP9 in vivo

Structural approaches:

• Cryo-electron microscopy: To visualize NOP9 within pre-ribosomal particles

• Cross-linking mass spectrometry (XL-MS): To map interaction interfaces at amino acid resolution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To identify regions that change conformation upon binding to partners

Genetic approaches:

• Synthetic genetic interaction screening: Identifying genes that show genetic interactions with NOP9 mutations

• Suppressor screening: Identifying mutations that suppress NOP9 conditional phenotypes

• CRISPR interference screens: Systematic identification of genes that modify NOP9-related phenotypes

Functional validation:

• In vitro reconstitution: Assembling purified components to recapitulate processing steps

• Single-molecule approaches: Fluorescence techniques to observe dynamics of interactions

• Complementation assays: Testing the ability of mutant versions to restore function

These approaches would generate a comprehensive interaction map, facilitating a systems-level understanding of NOP9's role in ribosome biogenesis.

How can researchers address the contradiction between NOP9's essential function and its potential as an antifungal target?

This apparent contradiction presents both challenges and opportunities:

Target validation approaches:

• Developing conditional mutants (e.g., temperature-sensitive alleles) to verify effects on virulence

• Using partial inhibition models to determine the threshold of NOP9 activity needed for viability

• Testing NOP9 depletion effects in different infection models to establish relevance to pathogenesis

Selective inhibition strategies:

• Detailed comparative analysis of fungal versus human NOP9 homologs

• Structure-based design of inhibitors that exploit species-specific differences

• Targeting fungal-specific interaction partners rather than NOP9 directly

Exploitation of species-specific contexts:

• C. glabrata may have unique dependencies on NOP9 function under host conditions

• Stress conditions during infection may create synthetic vulnerabilities

• Host factors might enhance the effect of partial NOP9 inhibition

Experimental design considerations:

• Testing inhibitors under conditions that mimic the host environment

• Combining NOP9 targeting with existing antifungals to achieve synergistic effects

• Developing fungal-specific delivery mechanisms to increase local drug concentration

By addressing these aspects methodically, researchers can resolve the contradiction and determine whether NOP9 represents a viable antifungal target despite its essential nature.