Recombinant Candida glabrata Cytochrome c oxidase assembly protein COX16, mitochondrial (COX16)

What is the subcellular localization of COX16 in Candida glabrata and how does it compare to human COX16?

Based on comparative analysis with human COX16, the C. glabrata COX16 protein is likely an inner mitochondrial membrane protein with its C-terminus facing the intermembrane space. In humans, COX16 lacks a predictable N-terminal presequence and is inserted into the inner mitochondrial membrane with a single transmembrane domain . The protein is only accessible to protease treatment when the outer membrane is disrupted and shows resistance to carbonate extraction, confirming its membrane integration . When studying C. glabrata COX16 localization, researchers should employ:

• Subcellular fractionation with differential centrifugation

• Protease protection assays with isolated mitochondria

• Carbonate extraction to differentiate between membrane-integrated and peripheral proteins

• Immunofluorescence microscopy with COX16-specific antibodies or epitope-tagged recombinant proteins

For definitive localization confirmation, researchers should generate GFP-fusion proteins and compare colocalization with established mitochondrial markers.

What is the primary function of COX16 in the mitochondrial respiratory chain of fungi?

COX16 is required for cytochrome c oxidase (Complex IV) assembly in the mitochondrial respiratory chain. Research indicates that COX16 plays a specific role in the biogenesis of the COX2 subunit and facilitates its association with the COX1-containing assembly module . In humans, COX16 specifically:

• Interacts with newly synthesized COX2

• Is required for SCO1 (but not SCO2) association with COX2, implicating it in CuA site formation

• Facilitates COX2 association with MITRAC assembly intermediate containing COX1

For studying this function in C. glabrata, researchers should implement:

• COX16 knockout/knockdown experiments followed by cytochrome c oxidase activity assays

• Blue-Native PAGE to analyze assembly intermediates and respiratory chain complexes

• Co-immunoprecipitation studies to identify interaction partners

• In-gel activity staining to assess functional consequences of COX16 disruption

How is COX16 expression regulated in Candida glabrata during different growth conditions?

While the search results don't specifically address regulation of COX16 in C. glabrata, researchers investigating this question should employ:

• qRT-PCR analysis of COX16 mRNA levels under various conditions:

  • Different carbon sources (glucose, glycerol, lactate)

  • Aerobic vs. hypoxic conditions

  • Presence/absence of respiratory inhibitors

  • Copper-limited vs. copper-replete conditions

• Western blot analysis for protein levels under the same conditions

• Promoter analysis using reporter constructs to identify regulatory elements

• Chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the COX16 promoter

Human COX16 is highly expressed in tissues with high energy demands, particularly skeletal and cardiac muscle . In C. glabrata, researchers should examine if expression varies during different growth phases and stress conditions, especially under host-mimicking conditions.

What are the optimal methods for recombinant expression and purification of C. glabrata COX16?

For successful recombinant expression and purification of C. glabrata COX16:

• Expression system selection:

  • E. coli: Use BL21(DE3) strain with pET vectors containing a cleavable N-terminal tag (His6 or GST)

  • Yeast expression: S. cerevisiae or P. pastoris systems may provide better folding for mitochondrial proteins

  • Cell-free systems: Consider for difficult membrane proteins

• Optimization strategies:

  • Express without the predicted transmembrane domain for improved solubility

  • Use fusion partners (MBP, SUMO) to enhance solubility

  • Employ low induction temperatures (16-20°C) for proper folding

  • Include mild detergents for membrane protein solubilization (DDM, LDAO, or Triton X-100)

• Purification protocol:

  • Affinity chromatography using His-tag or GST-tag

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for final polishing

  • For membrane-integrated form, solubilize with 1% DDM followed by affinity purification

• Quality control assessments:

  • Circular dichroism (CD) to verify secondary structure

  • Dynamic light scattering (DLS) to confirm monodispersity

  • Thermal shift assays to assess stability

What approaches are most effective for studying COX16 function in C. glabrata?

Multiple complementary approaches should be employed to study COX16 function:

How can researchers effectively measure the impact of COX16 mutations on cytochrome c oxidase assembly in C. glabrata?

To assess COX16 mutation effects on cytochrome c oxidase assembly:

• Mutation design strategy:

  • Target conserved residues identified through sequence alignment with human COX16

  • Create pathogenic-mimicking mutations based on human disease variants

  • Perform systematic alanine scanning of predicted functional domains

• Assembly analysis methods:

  • Blue-Native PAGE to visualize assembly intermediates and mature complex IV

  • In-gel activity staining to assess functional complexes

  • Pulse-chase labeling of mitochondrially synthesized subunits to track assembly kinetics

  • Quantitative proteomics to measure stability of individual subunits

• Functional assessment techniques:

  • Spectrophotometric measurement of cytochrome c oxidase activity

  • ELISA-based quantification of assembled complex IV

  • High-resolution respirometry to measure oxygen consumption

  • Growth curve analysis under conditions requiring respiratory function

• Copper-related assessments:

  • Copper supplementation experiments to test rescue of assembly defects

  • Metal content analysis of purified complex IV using ICP-MS

  • Cu⁺ transfer assays between metallochaperones and COX2

How does the interaction between COX16 and copper chaperones differ between human and C. glabrata systems?

While specific data for C. glabrata is limited, researchers investigating this question should:

• Identify C. glabrata homologs of human copper chaperones:

  • SCO1 and SCO2 homologs

  • COA6 homolog

  • Other copper chaperones involved in COX assembly

• Perform comparative interaction studies:

  • Co-immunoprecipitation experiments with tagged versions of COX16 and copper chaperones

  • Surface plasmon resonance (SPR) to measure binding affinities

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

• Functional assessment:

  • Generate knockout/knockdown strains of individual copper chaperones

  • Perform copper supplementation experiments to assess rescue effects

  • Create chimeric proteins with domains swapped between human and C. glabrata to identify specificity determinants

Research in human cells has shown that COX16 is required for SCO1 (but not SCO2) association with COX2, and pathogenic mutations in SCO1 and COA6 impact their association with COX16 . Researchers should determine if these differential interactions are conserved in C. glabrata.

What is the molecular mechanism by which COX16 facilitates copper insertion into the CuA site of COX2?

This advanced research question requires sophisticated approaches:

• Structural analysis:

  • Cryo-EM structures of COX16-COX2-SCO1 complexes at different stages of copper insertion

  • NMR studies of domain interactions with isotopically labeled proteins

  • Computational molecular dynamics simulations of copper transfer pathways

• Copper transfer assays:

  • In vitro reconstitution of copper transfer with purified components

  • Use of copper-specific fluorescent probes to track transfer kinetics

  • Stopped-flow kinetic analysis of transfer reactions

• Mutational analysis:

  • Site-directed mutagenesis of predicted copper-binding residues

  • Creation of chimeric proteins between different species to identify critical domains

  • CRISPR-mediated base editing for precise residue modifications

• Redox state analysis:

  • Redox-sensitive fluorescent proteins fused to COX16 domains

  • Mass spectrometry to identify redox-modified residues during copper transfer

  • EPR spectroscopy to monitor copper oxidation states

Current research suggests that despite lacking a canonical copper-binding motif, COX16 may be involved in copper delivery to COX2 . The exact mechanism remains to be elucidated.

How does C. glabrata COX16 function under the unique oxidative stress conditions encountered during host infection?

This question addresses the pathogen-specific context:

• Infection-mimicking conditions for study:

  • Macrophage co-culture systems with wild-type and COX16-deficient C. glabrata

  • Growth in serum or other host-mimicking media with varying copper availability

  • Exposure to reactive oxygen and nitrogen species mimicking host immune response

• Stress response analysis:

  • Transcriptomic analysis of COX16 and related genes under host-relevant stresses

  • Proteomic analysis of COX16 interactome changes during stress

  • Measurement of total antioxidant capacity under stress conditions

  • Quantification of ROS production using appropriate fluorescent probes

• Mutant phenotyping:

  • Survival analysis of COX16-deficient strains in macrophages

  • Virulence assessment in animal models

  • Competitive growth assays between wild-type and mutant strains

• Adaptive responses:

  • Evolution experiments under host-mimicking conditions to identify compensatory mechanisms

  • Analysis of clinical isolates for COX16 sequence and expression variations

How conserved is COX16 function across fungal species, particularly between opportunistic pathogens like C. glabrata and C. albicans?

To address this evolutionary question:

• Comparative sequence analysis:

  • Multiple sequence alignment of COX16 from diverse fungal species

  • Phylogenetic analysis to identify evolutionary relationships

  • Domain conservation analysis across species

  • Identification of conserved motifs and residues

• Functional complementation studies:

  • Cross-species complementation experiments (e.g., C. glabrata COX16 in S. cerevisiae cox16Δ)

  • Domain-swapping experiments between different fungal COX16 proteins

  • Heterologous expression of C. glabrata COX16 in human COX16-knockout cells

• Comparative interaction studies:

  • Yeast two-hybrid screens with COX16 from different species

  • Affinity purification-mass spectrometry to compare interactomes

  • Structure prediction and modeling to identify conserved interaction surfaces

• Phenotypic analysis:

  • Comparative growth analysis of COX16 mutants across species

  • Respiratory capacity measurements in different fungal backgrounds

  • Stress tolerance assessments across species

Human COX16 does not complement the yeast mutant strain, suggesting functional divergence . Similar comparative studies between C. glabrata and other fungi would be informative.

What role might COX16 play in the microevolution of C. glabrata during recurrent infections and antifungal treatment?

This question bridges evolutionary biology and clinical microbiology:

• Clinical isolate analysis:

  • Sequencing of COX16 from sequential clinical isolates from patients with recurrent candidiasis

  • Expression analysis of COX16 in clinical isolates with different antifungal susceptibilities

  • Search for signatures of selection in COX16 sequences from diverse geographical locations

• Experimental evolution approaches:

  • Serial passage experiments under antifungal pressure

  • Selection for respiratory deficiency and analysis of COX16 mutations

  • Copper limitation as a selective pressure for COX16 adaptation

• Phenotypic consequences assessment:

  • Antifungal susceptibility testing of COX16 variants

  • Virulence assessment of evolved strains

  • Fitness cost measurement of adaptations

• Population genetics analysis:

  • Comparison of COX16 sequence across different C. glabrata sequence types

  • Identification of recombination events affecting COX16

  • Assessment of COX16 diversity in hospital settings

Can the unique features of C. glabrata COX16 be exploited for selective therapeutic targeting?

This translational research question requires:

• Comparative structural analysis:

  • Identification of structural differences between human and C. glabrata COX16

  • Virtual screening for compounds that selectively bind fungal COX16

  • Fragment-based drug discovery approaches targeting unique pockets

• Vulnerability assessment:

  • Synthetic lethality screens to identify genes that become essential in COX16-deficient backgrounds

  • Chemical genetic screens to find compounds with enhanced activity against COX16 mutants

  • Metabolic profiling to identify compensatory pathways that could be co-targeted

• Drug development considerations:

  • Assay development for high-throughput screening

  • Structure-activity relationship studies of hit compounds

  • Selectivity testing against human COX16 and mitochondrial function

• Combination approaches:

  • Testing synergy between COX16-targeting compounds and existing antifungals

  • Exploration of copper chelators or ionophores as COX16-dependent sensitizers

  • Investigation of mitochondrial stress inducers as potentiators

How does copper availability affect the importance of COX16 in C. glabrata pathogenesis?

This question bridges basic biology and clinical relevance:

• In vitro approaches:

  • Growth and cytochrome c oxidase activity assessment under copper-limited conditions

  • Competition assays between wild-type and COX16-deficient strains at varying copper concentrations

  • Transcriptomic and proteomic analysis of copper-dependent responses

• Infection models:

  • Mouse infection models with controlled copper diets

  • Ex vivo infection of human tissues with varying copper levels

  • Cell culture infection models with copper chelators or supplements

• Clinical correlations:

  • Analysis of COX16 function in clinical isolates from patients with different copper status

  • Correlation of copper levels in infection sites with C. glabrata fitness

  • Assessment of copper homeostasis genes in clinical isolates

• Therapeutic implications:

  • Testing copper ionophores as antifungal agents

  • Investigating copper chelation as an adjuvant therapy

  • Developing copper-dependent targeted therapies

Human studies have shown that copper supplementation increases COX activity and restores normal steady-state levels of COX subunits in COX16 knockout cells . This suggests copper-based therapeutic approaches might be effective against C. glabrata infections.

What are the major technical challenges in expressing and studying recombinant C. glabrata COX16, and how can they be overcome?

Researchers face several challenges when working with this protein:

• Membrane protein expression challenges:

  Challenge	Solution Approach	Expected Outcome
  Protein aggregation	Use mild detergents (DDM, LDAO); Express at low temperature (16-20°C)	Increased soluble protein yield
  Low expression levels	Optimize codon usage; Use strong inducible promoters; Test multiple expression hosts	2-5 fold increased expression
  Improper folding	Co-express with chaperones; Use eukaryotic expression systems	Higher percentage of correctly folded protein
  Toxicity to host	Use tight expression control; Express toxic domains separately	Reduced host growth inhibition

• Functional assay challenges:

  Challenge	Solution Approach	Technical Details
  Distinguishing direct vs. indirect effects	Complementation with mutant variants; Acute protein depletion systems	Use degron tags for rapid protein depletion
  Measuring transient interactions	Proximity labeling (BioID, APEX); Crosslinking MS	Optimize labeling time (10-30 min) for transient interactions
  Assembly intermediate isolation	Tandem affinity purification; Gradient centrifugation	Use digitonin (1%) for gentle complex solubilization
  Copper transfer quantification	Copper-specific fluorescent probes; ICP-MS	Maintain anaerobic conditions to prevent oxidation

• Implementation strategies:

  • Start with soluble domains for initial characterization

  • Use nanodiscs or amphipols for membrane protein stabilization

  • Develop split-reporter assays for interaction studies

  • Implement controlled proteolysis to identify stable domains

How can researchers effectively integrate data from different experimental approaches to build a comprehensive model of COX16 function in C. glabrata?

This methodological question addresses data integration:

• Multi-omics data integration:

  • Combine transcriptomics, proteomics, and metabolomics data using pathway analysis tools

  • Implement network analysis to identify functional modules

  • Use machine learning approaches to predict functional relationships

  • Develop computational models that integrate diverse data types

• Structural-functional correlation:

  • Map functional data onto structural models

  • Identify structure-function relationships through comparative analysis

  • Use molecular dynamics simulations to predict effects of mutations

  • Implement integrative structural biology approaches combining multiple data types

• Systems biology approaches:

  • Develop mathematical models of cytochrome c oxidase assembly

  • Implement flux balance analysis to predict metabolic consequences

  • Use agent-based modeling for assembly pathway simulation

  • Apply sensitivity analysis to identify critical parameters

• Visualization and database tools:

  • Develop specialized databases for COX assembly factors

  • Create interactive visualization tools for complex datasets

  • Implement standardized data formats for sharing between research groups

  • Utilize existing pathway databases with custom overlays for COX16-specific data

By integrating these approaches, researchers can develop a comprehensive understanding of COX16 function that accounts for its roles in both normal physiology and pathological conditions.

What emerging technologies could revolutionize our understanding of COX16 function in C. glabrata?

Looking forward, several cutting-edge technologies hold promise:

• Advanced imaging techniques:

  • Cryo-electron tomography of mitochondria to visualize COX16 in native context

  • Super-resolution microscopy (PALM/STORM) for dynamic assembly visualization

  • Correlative light and electron microscopy (CLEM) to track labeled assembly factors

  • Live-cell single-molecule tracking to monitor COX16 dynamics

• Genome engineering advances:

  • CRISPR base editing for precise single-nucleotide modifications

  • CRISPRi/CRISPRa for temporal control of gene expression

  • Prime editing for precise sequence modifications

  • Genome-wide synthetic genetic arrays for comprehensive interaction mapping

• Structural biology innovations:

  • AlphaFold2 and related AI tools for accurate structure prediction

  • Integrative structural biology combining multiple data sources

  • Time-resolved structural methods to capture assembly intermediates

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Single-cell approaches:

  • Single-cell transcriptomics to identify cell-to-cell variation

  • Single-cell proteomics for protein-level heterogeneity

  • Microfluidic approaches for single-cell phenotyping

  • Spatial transcriptomics to map gene expression in infection contexts

What are the most critical unanswered questions about COX16 function that future research should address?

Priority research areas include:

• Mechanistic questions:

  • What is the step-by-step mechanism of COX16-facilitated copper insertion?

  • How does COX16 coordinate with other assembly factors temporally?

  • What signals regulate COX16 activity under different conditions?

  • How does COX16 contribute to assembly quality control?

• Pathogenesis-related questions:

  • How does host copper sequestration affect COX16 function during infection?

  • Does COX16 contribute to antifungal resistance mechanisms?

  • Can COX16 function be targeted without affecting human mitochondria?

  • How does COX16 function change during host adaptation?

• Evolutionary questions:

  • Why has COX16 function diverged between yeast and humans?

  • What selective pressures shape COX16 evolution in pathogenic fungi?

  • How did the COX16-dependent assembly pathway evolve?

  • Are there species-specific interaction partners for COX16?

• Therapeutic implications:

  • Can COX16-dependent pathways be exploited for combination therapies?

  • Would targeting COX16 reduce the emergence of antifungal resistance?

  • Could modulating copper homeostasis enhance existing antifungals?

  • Is COX16 function critical for persistence during antifungal treatment?

By addressing these questions, future research will not only advance our understanding of fundamental mitochondrial biology but also potentially reveal new therapeutic approaches for Candida infections.