Recombinant Candida dubliniensis Cytochrome oxidase assembly protein 3, mitochondrial (COA3)

What is Cytochrome Oxidase Assembly Protein 3 (COA3) and what is its role in Candida dubliniensis?

COA3 (also known as CD36_71440) is a mitochondrial transmembrane protein responsible for cytochrome c oxidase (COX) protein complex assembly in Candida dubliniensis. As a mitochondrial assembly factor, it plays a crucial role in the proper formation and function of respiratory chain complex IV (cytochrome c oxidase), which is essential for aerobic respiration . In Candida dubliniensis, this protein consists of 88 amino acids and contributes to mitochondrial energy metabolism through its involvement in the electron transport chain. The full amino acid sequence is: MGKLVGAPKGHDRYRDPKTHQITPALYRVRAPFFWRNTIALFAVSSIPLAVYLYTFKKMGDDDLGDIPIPPISDEELQKLKLEYENQK .

How does Candida dubliniensis COA3 compare structurally and functionally to COA3 homologs in other species?

While specific comparative data for C. dubliniensis COA3 is limited, research suggests functional conservation across species with some structural variations. The 88-amino acid length of C. dubliniensis COA3 is similar to homologs in other fungi, though the exact homology percentages are not provided in the available data. Research in human cells has shown that COA3 (CCDC56) promotes mitochondrial fragmentation through DRP1 phosphorylation . This suggests that COA3 may have additional roles beyond cytochrome c oxidase assembly that could be conserved across species.

To study this:

• Perform sequence alignment analysis using tools like BLAST or Clustal Omega

• Generate phylogenetic trees using the aligned sequences

• Compare predicted secondary structures using tools like PSIPRED

• Conduct functional complementation assays by expressing C. dubliniensis COA3 in other species with COA3 deletions

What protein domains and structural features characterize Candida dubliniensis COA3?

Based on its amino acid sequence, C. dubliniensis COA3 contains characteristic features of a mitochondrial membrane protein:

• A hydrophobic transmembrane domain (evident in the sequence: FFWRNTIALFAVSSIPLAVYLYTFKK)

• A mitochondrial targeting sequence (likely at the N-terminus)

• Charged residues distributed throughout the protein (including lysine, arginine, aspartic acid residues)

The protein's relatively small size (88 amino acids) suggests it may function as part of a larger protein complex rather than independently. Structural prediction algorithms would likely identify 1-2 transmembrane helices that anchor the protein in the mitochondrial membrane.

What are the optimal conditions for expressing and purifying recombinant Candida dubliniensis COA3?

For optimal expression and purification of recombinant C. dubliniensis COA3:

Expression System:

• E. coli is the recommended heterologous expression system

• BL21(DE3) strain is preferred for mitochondrial proteins

• Expression vector with N-terminal His-tag facilitates purification

Expression Conditions:

• Induction with 0.5-1.0 mM IPTG

• Lower temperature induction (16-20°C) for 16-20 hours to enhance proper folding

• Supplementation with 5% glycerol in culture medium to stabilize the protein

Purification Protocol:

• Cell lysis using sonication in Tris/PBS-based buffer (pH 8.0)

• Immobilized metal affinity chromatography using Ni-NTA resin

• Elution with imidazole gradient (50-250 mM)

• Buffer exchange to remove imidazole

• Concentration and storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

Storage Recommendations:

• Store at -20°C/-80°C

• Add 5-50% glycerol (50% is optimal) for long-term storage

• Avoid repeated freeze-thaw cycles

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

How can I verify the identity and functional integrity of recombinant Candida dubliniensis COA3?

To verify identity and functional integrity:

Identity Verification:

• SDS-PAGE analysis (expected MW: ~10-12 kDa including His-tag)

• Western blot using anti-His antibodies

• Mass spectrometry for precise molecular weight determination

• N-terminal sequencing to confirm the first 10-15 amino acids

Functional Assessment:

• Circular dichroism to assess secondary structure

• In vitro binding assays with known interaction partners

• Complementation assays in COA3-deficient yeast models

• Activity assays measuring cytochrome c oxidase assembly

Purity Assessment:

• SDS-PAGE should show >90% purity as a single band

• Size-exclusion chromatography can detect aggregation or degradation products

What assays can be used to study the interaction between COA3 and other components of the cytochrome c oxidase assembly pathway?

Several methodologies can assess COA3 interactions:

In vitro Assays:

• Pull-down assays: Using purified His-tagged COA3 as bait to capture interacting proteins

• Surface Plasmon Resonance (SPR): To determine binding kinetics and affinity

• Isothermal Titration Calorimetry (ITC): For quantitative binding parameters

• Microscale Thermophoresis: To measure interactions under near-native conditions

In vivo Assays:

• Co-immunoprecipitation: Using anti-His antibodies to pull down COA3 and associated proteins

• Yeast two-hybrid: To screen for novel interacting partners

• Bimolecular Fluorescence Complementation (BiFC): To visualize interactions in living cells

• Proximity Labeling: Using BioID or APEX2 fusions to identify proximal proteins

Functional Assays:

• Cytochrome c oxidase activity assays: Measure COX activity in the presence/absence of COA3

• Mitochondrial respiration measurements: Using oxygen consumption rate as a readout

• Blue Native PAGE: To assess complex assembly states

How should I interpret changes in mitochondrial morphology and function when studying COA3 in fungal models?

When analyzing COA3's impact on mitochondrial dynamics:

Morphological Analysis:

• Mitochondrial fragmentation may indicate increased fission or decreased fusion

• Compare changes observed to findings in other systems (e.g., COA3 promotes mitochondrial fragmentation in human cells through DRP1 phosphorylation)

• Use confocal microscopy with mitochondrial markers (like MitoTracker) to quantify:

  • Number of mitochondria per cell

  • Average mitochondrial length

  • Degree of mitochondrial interconnectivity

  • Mitochondrial distribution within the cell

Functional Assessment:

• Respiratory capacity measurements using respirometry

• Complex IV activity assays to directly assess cytochrome c oxidase function

• ATP production measurement to evaluate bioenergetic consequences

• Reactive oxygen species (ROS) quantification to assess oxidative stress

Interpretation Framework:

When interpreting these changes, consider that COA3 may influence both cytochrome c oxidase assembly and mitochondrial dynamics through different mechanisms, potentially through DRP1 phosphorylation at Ser616, as observed in human cell studies .

How can I differentiate between direct and indirect effects of COA3 manipulation in experimental systems?

Distinguishing direct from indirect effects requires systematic controls and validation:

Experimental Approaches:

• Temporal analysis: Track changes over time to establish sequence of events

• Domain mutation studies: Create point mutations in functional domains to dissect specific activities

• Rescue experiments: Reintroduce wild-type or mutant COA3 in knockout systems

• Selective inhibitors: Target downstream pathways to block indirect effects

Validation Strategies:

• Direct binding assays: Confirm physical interactions with proposed targets

• Proximity labeling: Identify proteins in close spatial proximity to COA3

• In vitro reconstitution: Recreate observed effects with purified components

• Comparative studies: Contrast effects in different genetic backgrounds

Analytical Framework:

What statistical approaches are appropriate for analyzing COA3 experimental data across different fungal species?

When conducting comparative analyses:

Statistical Methods for Different Data Types:

• Expression level comparisons:

  • ANOVA with post-hoc tests for multi-species comparisons

  • Linear mixed models when accounting for experimental variables

  • False discovery rate correction for multiple comparisons

• Phenotypic correlations:

  • Pearson/Spearman correlation for continuous variables

  • Chi-square tests for categorical outcomes

  • Regression analysis to establish predictive relationships

• Survival/growth analyses:

  • Kaplan-Meier curves with log-rank tests

  • Cox proportional hazards models for multivariable analysis

Data Normalization Approaches:

• Standardize to internal controls within each species

• Use z-scores to compare across species

• Apply quantile normalization for comparing distributions

• Consider relative fold changes rather than absolute values

Reporting Framework:

How can CRISPR-Cas9 gene editing be optimized for studying COA3 function in Candida dubliniensis?

CRISPR-Cas9 optimization for C. dubliniensis COA3 studies:

Guide RNA Design:

• Target unique regions of COA3 to avoid off-target effects

• Design multiple gRNAs targeting different exons

• Use C. dubliniensis codon optimization for Cas9 expression

• Consider species-specific PAM sequence requirements

Delivery Methods:

• Electroporation of ribonucleoprotein complexes (Cas9 protein + gRNA)

• Integration of Cas9 expression cassette using established C. dubliniensis transformation techniques

• Transient expression systems to minimize genomic integration

Genome Editing Strategies:

• Knockout approaches:

  • Design repair templates with selectable markers

  • Screen transformants using PCR and sequencing

  • Verify protein loss by Western blotting

• Tagged variant generation:

  • Design repair templates with C-terminal or N-terminal tags

  • Maintain endogenous promoter to preserve expression levels

  • Validate tag function using immunofluorescence or pull-down assays

• Point mutations:

  • Design repair templates with specific amino acid changes

  • Include silent mutations to prevent re-cutting

  • Screen by restriction digestion or sequencing

Experimental Validation:

• Perform complementation studies to confirm phenotype specificity

• Analyze off-target effects through whole-genome sequencing

• Create isogenic strains to minimize background genetic variations

How can recombinant COA3 be used to develop inhibitors targeting mitochondrial function in pathogenic Candida species?

Development of COA3-targeting inhibitors:

Target Identification:

• Perform structural analysis to identify unique regions in C. dubliniensis COA3

• Map interaction surfaces with assembly partners

• Identify species-specific domains absent in human homologs

• Focus on regions essential for cytochrome c oxidase assembly

Screening Approaches:

• In vitro binding assays:

  • Surface Plasmon Resonance with immobilized COA3

  • Fluorescence polarization with labeled peptide fragments

  • Thermal shift assays to identify stabilizing compounds

• Functional screens:

  • Cytochrome c oxidase assembly assays

  • Oxygen consumption rate measurements

  • Growth inhibition in COA3-dependent conditions

Compound Optimization:

• Structure-activity relationship studies

• Medicinal chemistry optimization for:

  • Mitochondrial penetration

  • Selectivity for fungal over human COA3

  • Stability in physiological conditions

Validation Framework:

What experimental approaches can reveal the evolutionary conservation of COA3 function across fungal pathogens?

To investigate evolutionary conservation:

Comparative Genomic Approaches:

• Identify COA3 homologs across fungal species using BLASTP

• Perform multiple sequence alignment to identify conserved domains

• Calculate selection pressure (dN/dS ratios) across different regions

• Construct phylogenetic trees to visualize evolutionary relationships

Functional Complementation:

• Express COA3 from different species in a C. dubliniensis COA3 knockout

• Measure restoration of:

  • Cytochrome c oxidase assembly

  • Respiration capacity

  • Growth under respiratory conditions

  • Mitochondrial morphology

Domain Swapping Experiments:

• Create chimeric proteins with domains from different species

• Test functionality of each chimera

• Identify domains responsible for species-specific functions

Comparative Interactomics:

• Perform co-immunoprecipitation followed by mass spectrometry

• Compare interaction partners across species

• Identify conserved and divergent binding partners

Analysis Framework:

What are common challenges in achieving proper folding of recombinant Candida dubliniensis COA3 and how can they be addressed?

Common folding challenges and solutions:

Expression Challenges:

• Inclusion body formation:

  • Lower induction temperature (16-20°C)

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Co-express with chaperones (GroEL/GroES)

  • Use specialized E. coli strains (Origami, SHuffle)

• Low expression yield:

  • Optimize codon usage for E. coli

  • Try different promoter systems

  • Adjust media composition (TB or 2YT instead of LB)

  • Extend expression time at lower temperatures

Purification Challenges:

• Protein aggregation:

  • Include mild detergents (0.05-0.1% DDM or LMNG)

  • Add stabilizing agents (glycerol, arginine, trehalose)

  • Perform purification at 4°C

  • Avoid concentrating above critical concentration

• Improper disulfide formation:

  • Include reducing agents during lysis

  • Perform controlled refolding if necessary

  • Consider oxidized/reduced glutathione pairs during refolding

Verification Methods:

• Circular dichroism to assess secondary structure

• Size exclusion chromatography to evaluate oligomeric state

• Thermal shift assays to monitor stability

• Limited proteolysis to assess proper folding

Optimization Table:

How can I address inconsistent results when studying COA3's role in mitochondrial dynamics across different experimental systems?

Strategies for addressing inconsistencies:

Standardization Approaches:

• Genetic background:

  • Use isogenic strains for all comparisons

  • Complement knockouts with identical expression constructs

  • Control for strain-specific differences with multiple independent isolates

• Expression level control:

  • Quantify COA3 expression by qPCR and Western blot

  • Use inducible promoters for controlled expression

  • Create stable cell lines rather than transient transfections

• Environmental standardization:

  • Control growth conditions precisely (temperature, pH, media composition)

  • Standardize cell density and growth phase

  • Account for oxygen availability, which affects mitochondrial function

Analytical Considerations:

• Technical replication:

  • Perform statistical power analysis to determine sample size

  • Include biological and technical replicates

  • Blind analysis where possible

• Complementary approaches:

  • Validate findings with multiple methodologies

  • Compare results from genetic knockdown vs. chemical inhibition

  • Correlate in vitro with in vivo observations

Reconciliation Framework:

What are the key considerations when designing experiments to investigate the potential connection between COA3, mitochondrial fragmentation, and metabolic reprogramming in Candida dubliniensis?

Experimental design considerations based on learnings from human COA3 studies:

Hypothesis Framework:
Research in human cells shows COA3 promotes mitochondrial fragmentation via DRP1 phosphorylation and enhances glycolysis . Similar pathways may exist in C. dubliniensis.

Key Parameters to Measure:

• Mitochondrial dynamics:

  • DRP1 phosphorylation status (particularly at Ser616 equivalent)

  • Mitochondrial morphology using fluorescence microscopy

  • Distribution of mitochondrial fission/fusion proteins

• Metabolic profiling:

  • Oxygen consumption rate (OCR)

  • Extracellular acidification rate (ECAR)

  • Glucose uptake and lactate production

  • ATP levels in different COA3 expression contexts

• Molecular mechanisms:

  • DRP1 localization (cytoplasmic vs. mitochondrial)

  • Activities of OXPHOS complexes (I, II, III, IV, V)

  • Levels of glycolytic and TCA cycle intermediates

Experimental Design Table:

This experimental design builds on findings from human studies while adapting to fungal systems, allowing investigation of whether the COA3-mediated mitochondrial fragmentation and metabolic shift observed in human cancer cells has evolutionary conservation in fungal species.

What are the emerging areas of research for Candida dubliniensis COA3 and its potential applications in antifungal drug development?

Current research suggests several promising directions:

• Structure-based drug design: Determining the three-dimensional structure of C. dubliniensis COA3 could enable rational design of inhibitors that disrupt cytochrome c oxidase assembly

• Metabolic vulnerability targeting: The connection between COA3, mitochondrial dynamics, and metabolic reprogramming suggests potential for targeting metabolic dependencies

• Comparative studies across Candida species: Understanding differences in COA3 structure and function across pathogenic Candida species could reveal species-specific vulnerabilities

• Host-pathogen interaction studies: Investigating how COA3-mediated mitochondrial functions influence virulence and host immune responses