Recombinant Candida glabrata COP9 signalosome complex subunit 5 (RRI1)

Advanced Research Questions

• What are the optimal methods for recombinant expression and purification of Candida glabrata RRI1?

  For successful recombinant expression of C. glabrata RRI1, researchers should consider a multi-faceted approach:

  a) Expression System Selection:

  • E. coli systems: BL21(DE3) or Rosetta strains for initial attempts

  • Yeast systems: S. cerevisiae or Pichia pastoris for proper folding and post-translational modifications

  • Insect cell systems: Consider for complex eukaryotic proteins with specific folding requirements

  b) Vector Design Considerations:

  • Include affinity tags (His6, GST, or MBP) for efficient purification

  • Codon optimization for the chosen expression host

  • Incorporate protease cleavage sites for tag removal

  c) Optimization Protocol:

  Parameter	Recommended Range	Notes
  Temperature	16-30°C	Lower temperatures often improve solubility
  Induction	0.1-1.0 mM IPTG (bacterial)	Optimize concentration
  Media	LB, TB, or defined media	Test different formulations
  Co-expression	Chaperones, CSN partners	May improve folding/solubility

  d) Purification Strategy:

  • Multi-step approach combining affinity chromatography with size exclusion and/or ion exchange

  • Buffer optimization to maintain protein stability and activity

  • Activity assays to confirm functional protein isolation

  Based on molecular biology methods used for RRI1-related gene expression in C. glabrata, appropriate primers can be designed for cloning and expression vector construction .

• How can the deneddylation activity of recombinant RRI1/CSN5 be effectively assessed?

  To assess the deneddylation activity of recombinant RRI1/CSN5, researchers can employ several complementary approaches:

  a) In vitro Deneddylation Assay:

  • Prepare neddylated cullins (Cdc53p/Cul1) as substrates

  • Incubate with purified recombinant RRI1/CSN5

  • Monitor deneddylation by western blotting using antibodies against the cullin and/or Rub1p/NEDD8

  • Include positive controls (human CSN) and negative controls (catalytically inactive mutants)

  b) Complementation Assays:

  • Use cell lysates from RRI1-deficient strains showing accumulated Rub1p-modified Cdc53p

  • Add recombinant RRI1/CSN5 and assess restoration of deneddylation activity

  • Wild-type cell lysate and purified human CSN can complement the deneddylation defect in vitro

  c) Analysis Protocol for Cullin Modification States:

  Step	Procedure	Expected Outcome
  1	Prepare total cell lysates from wild-type and Δrri1 strains	Two Cdc53p forms in wild-type, single modified form in mutant
  2	Perform SDS-PAGE and immunoblotting	Visualization of mobility shift
  3	Add recombinant RRI1/CSN5 to mutant lysate	Restoration of unmodified Cdc53p band
  4	Quantify band intensity ratios	Determination of deneddylation efficiency

  Wild-type cells display two forms of Cdc53p with different mobilities on SDS gels, while csn mutant strains accumulate Cdc53p exclusively in the Rub1p-modified form . This provides a clear readout for activity assessment.

• What experimental models are most appropriate for studying RRI1's role in pathogenesis?

  For studying RRI1's role in C. glabrata pathogenesis, several experimental models can be employed:

  a) Galleria mellonella (Wax Moth) Larvae Model:

  • This model has been successfully used for C. glabrata virulence studies and provides several advantages :

    • Ethical considerations and cost-effectiveness

    • Can be maintained at 37°C (human physiological temperature)

    • Possesses an innate immune system with strong similarity to the mammalian system

  • Methodology: Standardized inoculum injection, survival rate monitoring, hemolymph recovery at different timepoints to quantify fungal burden

  b) Macrophage Interaction Assays:

  • C. glabrata can survive and replicate within macrophages for prolonged periods

  • Protocol parameters:

  Parameter	Details	Measurements
  Cell types	J774.A1, RAW264.7, primary macrophages	Phagocytosis rate, intracellular survival
  MOI	1:1 to 10:1 (fungi:macrophage)	Optimize based on experimental goals
  Time points	1h, 24h, 48h	Match infection progression
  Analysis	Microscopy, CFU counting, cytokine profiling	Multi-parameter assessment

  c) Stress Response Assays:

  • Since RRI1 may affect stress responses like other virulence factors, assess:

    • Oxidative stress (H2O2, menadione)

    • Weak acid stress (acetic acid)

    • Nitrosative stress (NO donors)

  • Measure growth inhibition, gene expression changes, and protein modifications

• How can genome editing approaches be utilized to study RRI1 function in Candida glabrata?

  Modern genome editing approaches offer powerful tools for studying RRI1 function:

  a) CRISPR-Cas9 System Adaptation for C. glabrata:

  • Design sgRNAs targeting RRI1 gene regions

  • Construct repair templates for precise modifications

  • Deliver components via transformation

  b) Targeted Modifications:

  Modification Type	Application	Analysis Method
  Complete gene deletion	Null phenotype	Cullin modification analysis
  Domain-specific mutations	Structure-function	Biochemical activity assays
  Promoter replacement	Conditional expression	qRT-PCR, western blotting
  Epitope tagging	Localization, interactions	Immunofluorescence, AP-MS

  c) Phenotypic Characterization Pipeline:

  • Growth under various conditions (temperature, pH, carbon sources)

  • Stress tolerance profiles (oxidative, osmotic, cell wall stressors)

  • Virulence factor expression and secretion

  • Host cell interaction assays

  d) Complementation Strategy:

  • Reintroduce wild-type or mutant RRI1 variants

  • Assess restoration of normal cullin modification patterns

  • Compare deneddylation activity in vitro and in vivo

• How can contradictory data about RRI1 function be reconciled in experimental design?

  When facing contradictory data about RRI1 function, researchers should implement a systematic reconciliation approach:

  a) Strain and Condition Analysis:

  • Test RRI1 function across multiple C. glabrata strain backgrounds

  • Examine function under diverse conditions (media composition, temperature, pH)

  • Investigate genetic interaction context that might mask or reveal RRI1 functions

  b) Methodological Framework:

  Approach	Implementation	Benefit
  Method diversification	Use complementary techniques	Reduces method-specific artifacts
  Temporal resolution	Time-course experiments	Captures dynamic processes
  Quantitative analysis	Statistical rigor, replication	Increases confidence in results
  Comprehensive controls	Positive/negative controls	Validates experimental system

  c) Reconciliation Strategies:

  • Perform epistasis analysis to position contradictory functions in pathways

  • Use conditional systems to separate temporal roles

  • Develop mathematical models to integrate seemingly contradictory data

  • Employ single-cell approaches to identify population heterogeneity effects

• What techniques are most effective for studying protein-protein interactions of RRI1/CSN5?

  For comprehensive analysis of RRI1/CSN5 protein interactions:

  a) Affinity Purification-Mass Spectrometry (AP-MS):

  • Tandem affinity purification has successfully identified Rri1p/Csn5p-interacting proteins

  • TAP-tagging of RRI1/CSN5 followed by native condition purification

  • MS identification of co-purifying proteins

  • Quantitative approaches (SILAC, TMT labeling) to distinguish specific interactions

  b) Complementary Interaction Technologies:

  Technique	Application	Strengths
  Yeast Two-Hybrid	Binary interaction screening	High throughput, in vivo
  BioID/TurboID	Proximity-based labeling	Captures transient interactions
  Co-IP	Endogenous complex isolation	Preserves native interactions
  FRET/BiFC	Live-cell visualization	Spatial and temporal resolution

  c) Network Analysis:

  • Integrate interaction data from multiple sources

  • Identify core complex components versus transient interactors

  • Map interaction changes under different conditions (stress, growth phase)

  • Compare with interaction networks from related species

• What are the current challenges and future directions in studying the COP9 signalosome in pathogenic fungi?

  Current challenges and future research directions include:

  a) Structural and Compositional Challenges:

  • Limited homology between fungal and metazoan CSN subunits complicates comparative analysis

  • Uncertainty in assigning individual subunits to metazoan CSN1-8 counterparts

  • Need for improved structural biology approaches focused on fungal CSN complexes

  b) Research Frontiers:

  Challenge	Approach	Potential Impact
  Functional redundancy	Combinatorial gene deletion	Overcome compensatory mechanisms
  Pathogenicity connections	Host-pathogen interaction models	Identify virulence mechanisms
  Species variation	Comparative genomics	Universal vs. species-specific functions
  Integration with other systems	Multi-omics approaches	Comprehensive pathway mapping

  c) Therapeutic Implications:

  • Identification of pathogen-specific aspects of CSN function

  • Development of selective inhibitors of fungal CSN activity

  • Validation of CSN components as potential drug targets

  • Design of combination strategies targeting CSN-dependent pathways