Recombinant Candida glabrata Acyl-protein thioesterase 1 (CAGL0D02398g)

What is the functional role of Candida glabrata Acyl-protein thioesterase 1 (CAGL0D02398g) in cellular physiology?

Candida glabrata Acyl-protein thioesterase 1 (CAGL0D02398g) likely functions similarly to other acyl-protein thioesterases by catalyzing the deacylation of S-acylated peripheral membrane proteins. This enzymatic activity is crucial for maintaining dynamic protein localization and function. Based on studies of similar thioesterases, CAGL0D02398g likely participates in an acylation/deacylation cycle necessary for the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins in C. glabrata .

To investigate its functional role:

• Generate knockout strains using CRISPR-Cas9 gene editing

• Perform comparative proteomics to identify changes in the S-acylated proteome

• Conduct subcellular localization studies of known acylated proteins in wild-type versus knockout strains

• Analyze phenotypic changes related to virulence, stress response, and morphology

What expression systems are optimal for producing recombinant CAGL0D02398g?

The optimal expression system depends on your experimental goals. Based on research with similar proteins, consider these approaches:

For functional studies, S. cerevisiae expression may provide the most physiologically relevant protein, particularly when studying substrate specificity and regulatory mechanisms.

How is CAGL0D02398g regulated by the mating signaling pathway in C. glabrata?

The regulation of CAGL0D02398g may parallel other C. glabrata proteins involved in inter-species interactions. Research indicates that some C. glabrata proteins are regulated through the mating MAPK signaling pathway despite C. glabrata's predominantly asexual reproduction .

To investigate this regulatory relationship:

• Analyze the promoter region of CAGL0D02398g for binding sites of transcription factors downstream of the MAPK pathway

• Generate knockouts of key MAPK components (e.g., CgFus3, CgKss1) and measure CAGL0D02398g expression levels

• Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the CAGL0D02398g promoter

• Use reporter assays to measure promoter activity under various conditions that activate/inhibit the MAPK pathway

Recent findings suggest that the mating MAPK pathway in C. glabrata has been repurposed for inter-species communication, potentially involving deacylation enzymes like CAGL0D02398g in regulating the acylation status of secreted factors .

What role might CAGL0D02398g play in C. glabrata's interaction with C. albicans during mixed-species infections?

In mixed-species candidiasis, C. glabrata relies on C. albicans for efficient host colonization. Research has identified a novel C. glabrata protein (Yhi1) that facilitates this interaction by inducing hyphal growth in C. albicans . CAGL0D02398g may similarly contribute to inter-species interactions by regulating the acylation status of proteins involved in this process.

Methodological approach to investigate this role:

• Perform co-culture experiments with wild-type and CAGL0D02398g knockout C. glabrata strains with C. albicans

• Analyze the secretome of both species during co-culture using mass spectrometry

• Identify differentially acylated proteins in the presence/absence of CAGL0D02398g

• Test the effect of CAGL0D02398g overexpression on C. albicans morphology and virulence factor expression

How does substrate specificity of CAGL0D02398g compare to human acyl-protein thioesterases?

Understanding substrate specificity is crucial for developing selective inhibitors. To characterize CAGL0D02398g substrate preferences:

• Express and purify recombinant enzyme

• Test activity against a panel of acylated peptide substrates with varying:

  • Acyl chain lengths (C8-C20)

  • Degrees of unsaturation

  • Amino acid sequences flanking the acylated cysteine

• Compare kinetic parameters with human APT-1 and APT-2 using the same substrates

• Identify structural features contributing to specificity differences

Representative example of substrate specificity comparison:

*Note: This table presents hypothetical data based on typical values for similar enzymes and is intended as a methodological example.

What methods can be used to measure CAGL0D02398g enzymatic activity in vitro?

Multiple complementary approaches can be employed to measure CAGL0D02398g activity:

• Fluorogenic substrate assay:

  • Substrate: 4-methylumbelliferyl palmitate or similar fluorogenic substrates

  • Detection: Measure fluorescence increase upon substrate hydrolysis

  • Advantages: High-throughput, real-time monitoring

  • Limitations: Artificial substrate may not reflect physiological specificity

• Acylated peptide assay:

  • Substrate: Synthetic peptides with palmitoylated cysteines

  • Detection: HPLC or mass spectrometry to quantify deacylated products

  • Advantages: More physiologically relevant substrates

  • Protocol: Incubate enzyme with substrate, stop reaction at various timepoints, analyze products

• Radiolabeled substrate assay:

  • Substrate: [³H]-palmitoylated proteins or peptides

  • Detection: Measure release of [³H]-palmitate

  • Advantages: High sensitivity, suitable for kinetic studies

  • Limitations: Safety concerns, specialized facilities required

How can researchers identify the physiological substrates of CAGL0D02398g?

A multi-faceted approach is recommended:

• Acyl-biotin exchange (ABE) proteomics:

  • Compare wild-type and CAGL0D02398g knockout strains

  • Protocol:
    a. Block free thiols with N-ethylmaleimide
    b. Cleave thioester bonds with hydroxylamine
    c. Label newly exposed thiols with biotin-HPDP
    d. Enrich biotinylated proteins and analyze by mass spectrometry

  • Outcome: Proteins with increased acylation in knockout strains are potential substrates

• Proximity-based labeling:

  • Generate CAGL0D02398g fusions with BioID or APEX2

  • Express in C. glabrata and identify labeled proteins

  • Advantages: Identifies transient interactions in native cellular environment

• In vitro validation:

  • Express and purify candidate substrates

  • Perform in vitro deacylation assays with purified CAGL0D02398g

  • Confirm specificity through site-directed mutagenesis of catalytic residues

What are the optimal conditions for analyzing CAGL0D02398g localization and dynamics in living cells?

To study CAGL0D02398g localization and dynamics:

• Fluorescent protein tagging:

  • Generate C-terminal or N-terminal GFP/mCherry fusions

  • Validate functionality of tagged protein through complementation assays

  • Perform live-cell imaging using confocal microscopy

• Experimental considerations:

  • Temperature control: Maintain 30°C during imaging for physiological relevance

  • Time-lapse imaging: Capture images every 5-10 minutes for up to 2 hours

  • Co-localization: Use established organelle markers (ER, Golgi, plasma membrane)

  • Photobleaching techniques: FRAP or photoactivation to measure protein dynamics

• Protein dynamics during environmental changes:

  • Monitor localization changes during:
    a. Oxidative stress (0.5-2 mM H₂O₂)
    b. Temperature shift (30°C to 37°C)
    c. Antifungal treatment (sub-MIC concentrations)
    d. Co-culture with C. albicans

How should researchers interpret contradictory results when studying CAGL0D02398g function?

When faced with conflicting data:

• Examine experimental conditions:

  • pH and buffer composition significantly affect thioesterase activity

  • Reducing agents may influence enzyme conformation and activity

  • Temperature affects both enzyme kinetics and substrate accessibility

• Consider genetic background effects:

  • Different C. glabrata strains may show variable phenotypes

  • Compensatory mechanisms may mask knockout effects in certain strains

  • Strain-specific genetic interactions may influence results

• Evaluate cellular context:

  • In vitro versus in vivo discrepancies may reflect missing cofactors

  • Cell-type specific effects similar to those observed with APT-1 expression patterns

  • Growth phase and nutrient availability affect enzyme expression and activity

• Methodological troubleshooting matrix:

What bioinformatic approaches can identify potential regulatory elements in the CAGL0D02398g gene?

A comprehensive bioinformatic analysis should include:

• Promoter analysis:

  • Identify transcription factor binding sites using JASPAR, TRANSFAC databases

  • Focus on MAPK-responsive elements based on known regulation patterns

  • Compare with promoters of genes co-regulated during stress or infection

• RNA structure prediction:

  • Analyze 5' and 3' UTRs for regulatory RNA structures

  • Identify potential miRNA binding sites

  • Predict RNA-binding protein interaction motifs

• Comparative genomics:

  • Align regulatory regions across Candida species

  • Identify conserved non-coding sequences as potential functional elements

  • Compare with related thioesterases to identify common regulatory patterns

• Epigenetic analysis:

  • Analyze ChIP-seq data for histone modifications at the CAGL0D02398g locus

  • Identify potential DNA methylation sites

  • Map nucleosome positioning in the promoter region

How might CAGL0D02398g be targeted for novel antifungal development?

Recent research suggests that targeting protein deacylation pathways could yield novel antifungals . For CAGL0D02398g:

• Structure-based inhibitor design:

  • Generate high-resolution structures through X-ray crystallography or cryo-EM

  • Identify unique binding pockets absent in human homologs

  • Design competitive inhibitors that mimic transition states

  • Validate using enzyme assays and cell-based studies

• Peptide-based inhibitors:

  • Design peptides containing the pentapeptide motif identified in related research

  • Optimize using structure-activity relationship studies

  • Test efficacy against various Candida species in vitro and in vivo

• Potential development pathway:

  • Initial screening: Test compound libraries against purified enzyme

  • Secondary screening: Cell-based assays in C. glabrata

  • Lead optimization: Improve potency, selectivity, and pharmacokinetics

  • Preclinical testing: Animal models of Candida infection

What role might CAGL0D02398g play in biofilm formation and antifungal resistance?

Biofilm formation represents a major virulence factor and contributes to antifungal resistance. To investigate CAGL0D02398g's role:

• Biofilm assays:

  • Compare biofilm formation between wild-type and CAGL0D02398g knockout strains

  • Quantify using crystal violet staining, confocal microscopy, and metabolic assays

  • Analyze extracellular matrix composition and structure

• Mixed-species biofilms:

  • Evaluate C. glabrata-C. albicans mixed biofilms with and without CAGL0D02398g

  • Assess spatial organization using fluorescently labeled strains

  • Measure species-specific contributions to biomass and matrix

• Antifungal susceptibility:

  • Determine minimum inhibitory concentrations (MICs) for planktonic and biofilm cells

  • Test multiple antifungal classes (azoles, echinocandins, polyenes)

  • Measure persister cell formation in CAGL0D02398g mutants

• Mechanistic studies:

  • Identify acylated adhesins regulated by CAGL0D02398g

  • Measure cell surface hydrophobicity changes in mutants

  • Analyze transcriptional changes in biofilm-related genes

How does CAGL0D02398g contribute to C. glabrata adaptation to host environments?

Understanding the role of CAGL0D02398g in host adaptation requires:

• Infection models:

  • Murine systemic candidiasis model comparing wild-type and knockout strains

  • Tissue-specific colonization assays (kidney, liver, spleen)

  • Ex vivo macrophage interaction studies measuring phagocytosis and survival

• Stress response profiling:

  • Test growth under conditions mimicking host environments:
    a. Oxidative stress (H₂O₂, menadione)
    b. Nitrosative stress (GSNO)
    c. Nutrient limitation (carbon, nitrogen, iron)
    d. pH variation (pH 4-8)

  • Measure CAGL0D02398g expression under these conditions

• Host-pathogen protein interaction studies:

  • Identify host proteins interacting with CAGL0D02398g substrates

  • Analyze how deacylation affects these interactions

  • Determine impact on immune recognition and evasion

What controls are essential when performing deacylation assays with CAGL0D02398g?

Rigorous controls are critical for reliable deacylation assays:

• Enzyme controls:

  • Catalytically inactive mutant (mutation in the catalytic triad)

  • Heat-inactivated enzyme (95°C for 5 minutes)

  • Related thioesterase (e.g., human APT-1 or APT-2) for specificity comparison

• Substrate controls:

  • Non-acylated version of the same peptide/protein

  • Substrate with non-hydrolyzable acyl analog

  • Different acyl chain length variants to assess specificity

• Assay condition controls:

  • Enzyme concentration titration to ensure linear range

  • Time course to determine initial velocity conditions

  • Buffer-only reactions to establish background hydrolysis rates

• Inhibitor validation:

  • Known thioesterase inhibitors (e.g., palmostatin B) as positive controls

  • Structurally related non-inhibitory compounds as negative controls

  • Concentration-response curves to determine IC₅₀ values

How can researchers effectively study the impact of post-translational modifications on CAGL0D02398g activity?

To investigate how post-translational modifications affect CAGL0D02398g:

• Phosphorylation analysis:

  • Identify potential phosphorylation sites using prediction algorithms

  • Generate phosphomimetic (Ser/Thr to Asp/Glu) and phosphodeficient (Ser/Thr to Ala) mutants

  • Compare enzymatic activity and localization of mutants

  • Use Phos-tag gels to detect phosphorylated forms in vivo

• Mass spectrometry approaches:

  • Immunoprecipitate CAGL0D02398g from cells under different conditions

  • Perform LC-MS/MS analysis to identify and quantify modifications

  • Compare modification patterns during stress, host interaction, or drug treatment

• Site-directed mutagenesis:

  • Systematically mutate modified residues

  • Assess effects on:
    a. Enzyme activity using in vitro assays
    b. Protein stability through pulse-chase experiments
    c. Subcellular localization via fluorescence microscopy
    d. Protein-protein interactions via co-immunoprecipitation

What approaches can determine if CAGL0D02398g functions in multi-protein complexes?

To investigate protein complex formation:

• Co-immunoprecipitation studies:

  • Express epitope-tagged CAGL0D02398g in C. glabrata

  • Perform pull-downs under native conditions

  • Identify interacting partners using mass spectrometry

  • Validate interactions using reciprocal co-IPs

• Size exclusion chromatography:

  • Analyze native protein extracts or purified components

  • Compare elution profiles with size standards

  • Identify fractions containing CAGL0D02398g activity

  • Analyze composition of active fractions by western blotting or mass spectrometry

• Proximity-based approaches:

  • Implement split-reporter systems (BiFC, FRET) to visualize interactions in vivo

  • Use crosslinking mass spectrometry to capture transient interactions

  • Apply BioID or APEX2 proximity labeling to identify neighboring proteins

What are the most promising applications of CAGL0D02398g research for clinical mycology?

Research on CAGL0D02398g has several potential clinical applications:

• Diagnostic biomarker development:

  • Design antibodies or aptamers specific to CAGL0D02398g

  • Develop rapid detection methods for C. glabrata in mixed infections

  • Create assays that distinguish drug-resistant from susceptible strains

• Novel therapeutic strategies:

  • Target-based drug design focusing on CAGL0D02398g inhibition

  • Synthetic peptide derivatives similar to those identified in related research

  • Combination therapies targeting both acylation and deacylation pathways

• Virulence prediction:

  • Correlate CAGL0D02398g expression or polymorphisms with clinical outcomes

  • Develop molecular tests to predict virulence potential of clinical isolates

  • Guide personalized antifungal therapy based on molecular profiling

How does current understanding of CAGL0D02398g compare with knowledge of thioesterases in other pathogenic fungi?

Comparative analysis reveals:

• Evolutionary conservation:

  • Thioesterases are conserved across pathogenic fungi but show species-specific adaptations

  • CAGL0D02398g likely represents a specialized adaptation in C. glabrata

  • Similar enzymes in other Candida species may have divergent functions

• Functional differences:

  • C. albicans thioesterases have been more extensively studied

  • Unique aspects of C. glabrata biology (asexual reproduction, extreme stress resistance) may influence CAGL0D02398g function

  • Different substrate preferences reflect evolutionary adaptations to host niches

• Research gaps:

  • Limited structural information across fungal thioesterases

  • Incomplete understanding of regulatory mechanisms

  • Need for comprehensive substrate identification

• Future directions:

  • Comparative functional genomics across Candida species

  • Investigation of thioesterase roles in emerging pathogenic fungi

  • Systems biology approaches to model protein acylation/deacylation networks