Recombinant Candida glabrata ATP-dependent rRNA helicase SPB4 (SPB4), partial

What is the function of ATP-dependent rRNA helicase SPB4 in Candida glabrata?

SPB4 in C. glabrata functions as an ATP-dependent RNA helicase involved in ribosome biogenesis, specifically in the synthesis of 60S ribosomal subunits. Based on homology with S. cerevisiae Spb4p, it plays a critical role in the processing of 27SB pre-rRNAs to produce mature 25S and 5.8S rRNAs. This processing step is essential for the assembly of functional ribosomes, which are fundamental for protein synthesis and cellular growth.

When studying SPB4 function, researchers should consider employing genetic depletion strategies (similar to those used for S. cerevisiae Spb4p) to observe the resultant phenotypes. Analyzing pre-rRNA processing through pulse-chase labeling, northern hybridization, and primer extension can provide valuable insights into the specific steps affected by SPB4 depletion .

What is the subcellular localization of SPB4 in C. glabrata?

While specific localization data for C. glabrata SPB4 is limited in the current literature, inferences can be made based on its S. cerevisiae homolog. In S. cerevisiae, Spb4p is predominantly localized to the nucleolus and adjacent nucleoplasmic areas, consistent with its role in ribosome biogenesis. On sucrose gradients, it is found almost exclusively in rapidly sedimenting complexes and shows a peak in fractions containing the 66S pre-ribosomes .

To determine SPB4 localization in C. glabrata experimentally, researchers should consider:

• Creating a GFP or epitope-tagged version of SPB4 (similar to the HA epitope-tagged Spb4p used in S. cerevisiae research)

• Employing fluorescence microscopy for direct visualization

• Using subcellular fractionation followed by western blotting to identify the protein in specific cellular compartments

• Conducting sucrose gradient fractionation to evaluate association with pre-ribosomal particles

How does SPB4 compare to other RNA helicases in C. glabrata?

C. glabrata possesses multiple ATP-dependent RNA helicases, including SPB4 and DBP4, which likely have distinct but potentially overlapping functions in RNA metabolism. Based on available data, the following comparison can be made:

HAS1 (CAGL0M13519g) is another ATP-dependent RNA helicase involved in ribosome biogenesis in C. glabrata, as noted in acid stress response studies . Both comparative sequence analysis and functional studies would be necessary to fully characterize the functional relationships between these helicases.

What experimental approaches can be used to study SPB4's role in ribosome assembly?

To investigate SPB4's role in ribosome assembly, researchers should consider a multi-faceted approach:

• Genetic manipulation strategies:

  • Gene deletion using homologous recombination (challenging due to its essential nature)

  • Conditional expression systems (e.g., tetracycline-regulated promoters)

  • CRISPR-Cas9 for precise mutations in functional domains

• Ribosome profiling techniques:

  • Polysome profiling to assess 60S subunit levels and polysome formation

  • Sucrose gradient fractionation followed by northern blotting to track pre-rRNA processing

  • Mass spectrometry analysis of isolated pre-ribosomes to identify associated factors

• RNA analysis methods:

  • Pulse-chase labeling with [³H]uracil or [³²P]orthophosphate

  • Northern hybridization with probes specific for different pre-rRNA intermediates

  • Primer extension to map precise processing sites

• Protein interaction studies:

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

  • Yeast two-hybrid screening

  • Co-immunoprecipitation with known ribosome assembly factors

When interpreting results, researchers should analyze both direct effects (immediate consequences of SPB4 depletion) and indirect effects (later consequences that may affect earlier pre-rRNA processing steps), as observed with S. cerevisiae Spb4p .

How does SPB4 activity potentially respond to antifungal treatment and stress conditions?

While direct data on SPB4's response to antifungals is limited, research on C. glabrata's transcriptional response provides context for designing experiments to investigate this question. Based on studies of C. glabrata's response to macrophage phagocytosis and azole antifungals:

• Experimental design for investigating SPB4 during stress:

  • Create a tagged version of SPB4 (e.g., SPB4-3XFLAG) to monitor protein levels

  • Expose C. glabrata to various stressors (azoles, oxidative stress, hypoxia, pH stress)

  • Measure SPB4 expression through qRT-PCR and protein levels through Western blotting

  • Assess ribosome biogenesis efficiency under these conditions

• Relevant stress responses to consider:

  • Azole treatment induces expression of ergosterol biosynthesis genes through transcription factors like Zcf27

  • Hypoxic conditions trigger specific transcriptional responses, such as induction of Zcf4

  • pH stress activates transcription factors like Asg1p and Hal9p

Given that protein synthesis is energy-intensive, cells may regulate ribosome biogenesis factors like SPB4 during stress to conserve resources. Research on S. cerevisiae suggests ribosome biogenesis is downregulated during various stresses, but the specific regulation of SPB4 in C. glabrata requires direct investigation.

What is known about SPB4's potential role in C. glabrata pathogenicity?

While direct evidence linking SPB4 to C. glabrata pathogenicity is not available in the provided literature, several considerations suggest it may be indirectly important:

• Ribosome biogenesis and virulence:

  • Proper protein synthesis is essential for expression of virulence factors

  • Ribosome assembly factors have been implicated in virulence in other fungi

• Host adaptation mechanisms:

  • C. glabrata must adapt to various microenvironments within the host

  • Temporal transcriptional responses during macrophage infection involve specialized pathways activated chronologically

  • Factors involved in iron homeostasis, oxidative stress response, and metal ion sequestration show distinct expression patterns during infection

• Experimental approaches to investigate SPB4's role in pathogenicity:

  • Generate conditional SPB4 mutants and assess virulence in mouse models

  • Examine SPB4 expression during different stages of infection

  • Investigate whether SPB4 activity is modulated by host-relevant conditions (temperature, pH, oxidative stress)

  • Assess whether SPB4 inhibition affects known virulence traits

While ribosome biogenesis is a fundamental process, its components may be particularly important during the rapid adaptation required for pathogenesis and could potentially serve as targets for antifungal development.

What are the optimal storage and handling conditions for recombinant SPB4?

Based on the product information for recombinant Candida glabrata ATP-dependent rRNA helicase SPB4, the following storage and handling conditions are recommended:

• Long-term storage:

  • Store liquid form at -20°C/-80°C for up to 6 months

  • Store lyophilized form at -20°C/-80°C for up to 12 months

  • Add glycerol to a final concentration of 5-50% (default 50%) for long-term storage

• Working solutions:

  • Keep working aliquots at 4°C for up to one week

  • Repeated freezing and thawing is not recommended

• Reconstitution:

  • Briefly centrifuge vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Quality control considerations:

  • Purity should be >85% as determined by SDS-PAGE

  • Monitor activity after reconstitution with appropriate helicase assays

What methodological approaches can be used to assess the enzymatic activity of recombinant SPB4?

To evaluate the enzymatic activity of recombinant SPB4, researchers should consider the following methodological approaches:

• RNA unwinding assays:

  • Prepare double-stranded RNA substrates with one strand radioactively labeled

  • Incubate with SPB4 in the presence of ATP and reaction buffer

  • Analyze products by non-denaturing gel electrophoresis

  • Quantify unwinding activity by measuring the appearance of single-stranded RNA

• ATP hydrolysis assays:

  • Measure ATP hydrolysis using colorimetric phosphate detection methods

  • Compare ATPase activity in the presence and absence of RNA substrate

  • Determine kinetic parameters (Km, Vmax) under different conditions

• RNA binding assays:

  • Electrophoretic mobility shift assay (EMSA) with labeled RNA

  • Fluorescence anisotropy with fluorescently labeled RNA

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Buffer optimization considerations:

  • Test activity across pH range (typically 7.0-8.5)

  • Optimize Mg²⁺ concentration (typically 2-5 mM)

  • Determine optimal KCl/NaCl concentration (typically 50-150 mM)

  • Evaluate requirement for additional cofactors

• Substrate specificity assessment:

  • Test activity on different RNA structures (hairpins, duplexes)

  • Evaluate preference for specific RNA sequences

  • Determine whether SPB4 shows specificity for pre-rRNA substrates

How can I design experiments to specifically study SPB4's role in 27SB pre-rRNA processing?

To investigate SPB4's role in 27SB pre-rRNA processing in C. glabrata, consider the following experimental design:

How does C. glabrata SPB4 compare functionally to its homologs in S. cerevisiae and other fungi?

Based on the available data, we can draw the following comparisons between C. glabrata SPB4 and its homologs:

To experimentally establish functional equivalence between C. glabrata SPB4 and S. cerevisiae Spb4p, researchers could:

• Perform complementation experiments by expressing C. glabrata SPB4 in S. cerevisiae spb4 mutants to determine if it rescues the growth and ribosome biogenesis defects

• Create chimeric proteins with domains from both species' proteins to identify functionally important regions

• Conduct comparative structural modeling to identify conserved and divergent features that might relate to species-specific functions

• Perform comparative transcriptomics and proteomics analyses of SPB4/Spb4p-depleted cells to identify conserved and species-specific targets

How has SPB4 evolved in pathogenic versus non-pathogenic fungi, and what implications might this have?

While the search results don't provide direct information about SPB4 evolution across different fungi, researchers can address this question through:

• Phylogenetic analysis:

  • Construct phylogenetic trees of SPB4 homologs from diverse fungi

  • Compare evolutionary rates in pathogenic versus non-pathogenic lineages

  • Identify positively selected residues that might relate to pathogenicity

• Domain architecture comparison:

  • Analyze conservation of functional domains (DEAD-box, helicase domain)

  • Identify unique insertions or deletions in pathogenic fungi

  • Compare N- and C-terminal extensions that might confer specialized functions

• Expression pattern differences:

  • Compare expression regulation in response to host-like conditions

  • Analyze promoter regions for differences in regulatory elements

  • Evaluate expression during infection or exposure to antifungals

An interesting parallel can be drawn from the example of C. glabrata maintaining two Hap1 homologs (Zcf27 and Zcf4) that have evolved distinct roles allowing adaptation to specific host and environmental conditions . Similarly, SPB4 may have acquired specialized functions in pathogenic species that contribute to their survival in host environments.

What is the relationship between SPB4 and other RNA helicases involved in fungal ribosome biogenesis?

RNA helicases play crucial roles at various steps of ribosome biogenesis. From the available data, we can identify several RNA helicases involved in this process in C. glabrata:

• SPB4: Likely involved in 60S ribosomal subunit synthesis and processing of 27SB pre-rRNAs (based on S. cerevisiae homolog)

• DBP4: ATP-dependent RNA helicase (EC 3.6.4.13) that likely functions in pre-rRNA processing, potentially at a different step than SPB4

• HAS1: ATP-dependent RNA helicase involved in ribosome biogenesis, expression of which is altered during acid stress

To experimentally investigate the relationships between these helicases:

• Generate conditional mutants for each helicase and analyze the specific pre-rRNA processing steps affected

• Perform synthetic genetic interaction studies to identify functional relationships (synthetic lethality would suggest separate pathways, while suppression might indicate sequential action)

• Conduct biochemical purification of pre-ribosomal particles to determine which helicases are present in the same or different complexes

• Use RNA-seq to identify global effects of each helicase on the transcriptome and identify common or distinct targets

Understanding the division of labor between these helicases could provide insights into the coordination of the complex process of ribosome biogenesis and potentially reveal fungal-specific features that could be exploited for antifungal development.

How might SPB4 function intersect with stress response pathways in C. glabrata?

While direct evidence linking SPB4 to stress response in C. glabrata is limited, we can hypothesize potential intersections based on available data:

• Adaptation to host environments:

  • C. glabrata exhibits transcriptional responses to macrophage phagocytosis, with genes of specialized pathways activated chronologically at different infection times

  • Ribosome biogenesis, an energy-intensive process, likely requires regulation during stress adaptation

  • SPB4, as a factor in ribosome assembly, may be modulated to adjust protein synthesis capacity during stress

• Potential regulation mechanisms:

  • Transcriptional control: Expression patterns might change under different stresses

  • Post-translational modifications: Activity could be regulated by phosphorylation or other modifications

  • Protein-protein interactions: Association with different cofactors might redirect function

  • Localization changes: Relocalization could affect access to substrates

• Experimental approaches to investigate:

  • Analyze SPB4 expression and protein levels under various stresses (oxidative, pH, temperature, nutrient limitation)

  • Perform phosphoproteomics to identify potential regulatory modifications

  • Use BioID or proximity labeling to identify stress-specific interaction partners

  • Create reporter strains to monitor SPB4 localization during stress

The discovery that transcription factors like Asg1p and Hal9p regulate pH homeostasis and that Zcf27 and Zcf4 have evolved distinct roles for adaptation to specific environments suggests that C. glabrata employs sophisticated regulatory mechanisms to adapt to stress, which likely extend to the regulation of fundamental processes like ribosome biogenesis involving SPB4.

How can researchers distinguish between direct and indirect effects when studying SPB4 function?

Distinguishing direct from indirect effects is crucial when studying essential proteins like SPB4. Researchers should consider the following methodological approaches:

• Temporal analysis:

  • Use rapid depletion systems (e.g., auxin-inducible degron) to observe immediate consequences

  • Perform time-course experiments to distinguish primary from secondary effects

  • Compare early versus late transcriptional and proteome changes

• Direct biochemical approaches:

  • Use CLIP-seq (Cross-Linking Immunoprecipitation followed by sequencing) to identify direct RNA targets

  • Perform in vitro binding and unwinding assays with purified components

  • Use structure-guided mutagenesis to create separation-of-function mutants

• Comparative analysis:

  • Compare phenotypes with other factors known to act at the same or different steps

  • Use double mutant analysis to establish epistatic relationships

  • Analyze suppressor mutations that can bypass the requirement for SPB4

• Data integration strategies:

  • Combine different omics approaches (transcriptomics, proteomics, metabolomics)

  • Use network analysis to identify direct connections

  • Apply mathematical modeling to predict direct versus indirect effects

In S. cerevisiae, it was observed that Spb4p depletion initially affected processing of 27SB pre-rRNAs, but at later times also inhibited early pre-rRNA processing steps at sites A0, A1, and A2 . This demonstrates how secondary effects can emerge over time, emphasizing the importance of temporal analyses in distinguishing direct from indirect consequences.

What specialized techniques are required to study pre-ribosomal particle association of SPB4?

Studying pre-ribosomal particle association requires specialized techniques for isolating and analyzing these large, dynamic complexes. Researchers should consider:

• Particle isolation methods:

  • Sucrose gradient centrifugation to separate particles of different sizes

  • Affinity purification using tagged ribosomal proteins or assembly factors

  • Size exclusion chromatography for separation based on hydrodynamic radius

  • Glycerol gradient-density centrifugation combined with chemical crosslinking (GraFix) to stabilize complexes

• Composition analysis:

  • Mass spectrometry to identify protein components

  • RNA-seq for comprehensive RNA content analysis

  • Northern blotting with specific probes for pre-rRNA intermediates

  • Western blotting to detect specific proteins of interest

• Structural characterization:

  • Cryo-electron microscopy for structural determination

  • Chemical probing to analyze RNA structures

  • Crosslinking and footprinting to map protein-RNA interactions

  • Fluorescence microscopy to track particles in vivo

• Dynamic association studies:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure exchange rates

  • Single-particle tracking to observe dynamics in living cells

  • Pulse-chase experiments with tagged components to track assembly and disassembly

In S. cerevisiae, sucrose gradient fractionation showed that Spb4p was found almost exclusively in rapidly sedimenting complexes with a peak in fractions containing the 66S pre-ribosomes . Similar approaches would be valuable for studying C. glabrata SPB4, with the addition of modern techniques like proximity labeling and quantitative proteomics to provide a more comprehensive view of its interactions within pre-ribosomal particles.