Recombinant Candida glabrata Ubiquitin-conjugating enzyme E2 6 (UBC6), partial

What is Candida glabrata UBC6 and what distinguishes it from other E2 enzymes?

Candida glabrata UBC6 is a ubiquitin-conjugating (E2) enzyme that participates in the ubiquitination pathway, a crucial post-translational modification process. Unlike most E2 enzymes that form isopeptide bonds with lysine residues, UBC6 and its homologs are unique in their ability to mediate serine/threonine ubiquitination via hydroxyester linkages . Structurally, UBC6 lacks several conserved motifs and residues typically involved in ubiquitin transfer in other E2 enzymes. Instead, it contains a distinctive, highly conserved insertion near its active-site cysteine (C87), comprising serine (S89) and histidine (H94) residues that are critical for its unique catalytic function .

How does Candida glabrata UBC6 participate in protein quality control mechanisms?

Protein quality control via ubiquitination occurs through the sequential action of three enzyme classes: ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligases (E3) . As an E2 enzyme, UBC6 plays a central role in this cascade by receiving activated ubiquitin from E1 and catalyzing its transfer to target substrates. In C. glabrata, UBC6 contributes to protein quality control through non-canonical ubiquitination, facilitating the attachment of ubiquitin to hydroxylated amino acids (serine and threonine) via hydroxyester bonds . This distinctive mechanism expands the repertoire of protein modifications available for quality control and may be particularly relevant for membrane-associated proteins that undergo ER-associated degradation (ERAD).

How is UBC6 regulated in C. glabrata during stress conditions?

Adaptation to stress involves changes in global SUMOylation in C. glabrata , which may indirectly affect UBC6 activity. While direct regulation of UBC6 under stress conditions isn't fully characterized in the provided search results, we know that post-translational modifications are essential for C. glabrata's adaptation to stress and host environments. As an opportunistic pathogen, C. glabrata must adapt to various environmental conditions, including oxidative stress within host immune cells and nutrient limitations . The regulatory networks controlling UBC6 expression and activity likely respond to these stressors, potentially modulating protein degradation pathways to maintain cellular homeostasis under challenging conditions.

What structural features enable UBC6 to perform non-canonical ubiquitination?

UBC6 contains several unique structural features that facilitate non-canonical ubiquitination:

• A highly conserved insertion near the active-site cysteine (C87)

• Conserved serine (S89) residue within this insertion

• Critical histidine (H94) residue that imparts reactivity toward hydroxylated amino acids

Research demonstrates that H94 must exist in its neutral, non-protonated form to exert its activity, suggesting it functions as a general base in the catalytic mechanism . This histidine residue likely activates the hydroxyl groups of serine and threonine residues, enabling their nucleophilic attack on the UBC6~ubiquitin thioester intermediate. The positioning of these key residues in the three-dimensional structure creates a unique microenvironment that favors hydroxyester bond formation rather than the conventional isopeptide linkages catalyzed by other E2 enzymes.

How do the molecular dynamics of UBC6-ubiquitin interaction influence substrate selection?

The specific orientation of the ubiquitin C-terminus relative to the UBC6 active site likely influences substrate accessibility and selectivity. The presence of the conserved H94 residue creates a distinct electrostatic environment that may preferentially recognize and activate serine/threonine residues over lysine residues in target substrates.

What is the significance of the H94 residue in UBC6's catalytic mechanism?

H94 in UBC6 plays a critical role in enabling non-canonical ubiquitination. Experimental evidence indicates that H94 must be in its neutral, non-protonated form to facilitate ubiquitin transfer to hydroxylated amino acids . This suggests that H94 functions as a general base in the catalytic mechanism, activating the hydroxyl groups of serine and threonine residues to increase their nucleophilicity for attacking the UBC6~ubiquitin thioester bond.

The significance of H94 has been confirmed through substitution experiments using methylated histidine, which demonstrated that the protonation state of this residue is crucial for its catalytic activity . This mechanistic insight helps explain how UBC6 achieves specificity for non-canonical ubiquitination via hydroxyester linkages, distinguishing it from other E2 enzymes that primarily catalyze isopeptide bond formation with lysine residues.

What are the optimal methods for expressing and purifying recombinant C. glabrata UBC6?

Based on published research protocols, recombinant C. glabrata UBC6 can be effectively expressed and purified using the following approach:

• Expression system: E. coli cultured in LB medium, induced with 0.5 mM IPTG

• Growth conditions: 18°C overnight incubation

• Cell harvesting: Centrifugation at 4000 rpm for 15 minutes at 4°C

• Cell lysis: Resuspension in buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 30 mM Imidazole) followed by microfluidizer treatment (80 psi, two passages) and immediate addition of 1 mM PMSF

• Purification: Affinity chromatography followed by size exclusion chromatography using Superdex 200 increase 10/300 column equilibrated with 20 mM HEPES-NaOH, 250 mM NaCl, 200 mM Sucrose, 6 mM DM at pH 7.4

For studies requiring the UBC domain only, researchers typically use a construct containing amino acids 1-172 of UBC6 .

How can researchers effectively analyze UBC6-ubiquitin interactions using NMR spectroscopy?

NMR spectroscopy provides valuable insights into UBC6-ubiquitin interactions. A recommended protocol includes:

• Sample preparation: Production of 15N-enriched ubiquitin in isotope-enriched M9 minimal medium using 15NH4Cl as the nitrogen source

• Conjugate formation: Generation of disulfide-linked Ubc6-SS-Ub conjugates by incubating Ubc6 UBC WT (amino acids 1-172) or S89A variants containing a single cysteine residue with a threefold molar excess of disulfide-linked Ub(G76C)-TNB adduct for 1 hour at room temperature

• Purification: Ion exchange chromatography followed by SDS-PAGE analysis under non-reducing conditions to identify pure Ubc6 UBC-SS-Ub complexes

• NMR experiments: Two-dimensional [15N,1H]-HSQC and [15N,1H]-TRACT measurements with appropriate relaxation delays

• Data processing: Using NMRPipe for processing and CCPN for analysis

This approach allows researchers to examine structural changes and dynamics at the UBC6-ubiquitin interface, providing mechanistic insights into the catalytic process.

What methods are effective for studying UBC6-mediated ubiquitination in vitro?

To study UBC6-mediated ubiquitination in vitro, researchers can employ several complementary approaches:

• Fluorescence anisotropy assays: Labeling UBC6 with fluorescent maleimide dyes to monitor interactions with ubiquitin and substrate proteins

• Molecular dynamics simulations: Creating models of Ubc6-thioester loaded ubiquitin to study conformational changes during the catalytic cycle

• Site-directed mutagenesis: Generating variants (e.g., S89A, H94 modifications) to assess the functional importance of specific residues

• In vitro ubiquitination assays: Reconstituting the ubiquitination cascade with purified E1, UBC6 (E2), appropriate E3 ligases, ubiquitin, and target substrates

• Mass spectrometry: Identifying ubiquitination sites and quantifying the prevalence of different linkage types (isopeptide vs. hydroxyester)

These methods collectively enable detailed characterization of UBC6's catalytic mechanism, substrate specificity, and the kinetics of non-canonical ubiquitination reactions.

How does UBC6 contribute to C. glabrata pathogenicity and host adaptation?

While the direct connection between UBC6 and C. glabrata pathogenicity isn't explicitly detailed in the provided search results, we can infer potential relationships based on general principles of protein quality control and stress response:

• UBC6-mediated ubiquitination likely contributes to protein quality control mechanisms that maintain cellular homeostasis during host colonization

• As C. glabrata adapts to diverse environments within the human host, UBC6 may participate in remodeling the proteome to optimize survival and virulence

• The unique ability of UBC6 to form hydroxyester linkages expands the repertoire of regulatory modifications available to C. glabrata, potentially enhancing its adaptability

Research in related fungal pathogens indicates that post-translational modifications, including ubiquitination, play crucial roles in virulence and stress adaptation . The non-canonical ubiquitination mediated by UBC6 may represent an adaptive strategy that distinguishes C. glabrata from other Candida species and contributes to its success as an opportunistic pathogen.

What is the relationship between UBC6 and antifungal drug resistance mechanisms?

• Protein quality control mechanisms, including UBC6-mediated ubiquitination, may influence the stability and activity of drug efflux pumps that contribute to azole resistance

• UBC6 might participate in the degradation of misfolded proteins resulting from antifungal drug stress

• Stress adaptation pathways involving post-translational modifications are known to influence drug resistance in C. glabrata

C. glabrata exhibits reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis compared to other Candida species . While specific mutations in transcription factors like ROX1 and UPC2A have been implicated in fluconazole resistance , the potential role of UBC6 in these resistance mechanisms represents an interesting area for future research.

How does UBC6 interact with SUMOylation pathways in C. glabrata?

The interplay between ubiquitination and SUMOylation pathways represents an important regulatory network in C. glabrata:

• SUMOylation is essential for cell growth, division, and adaptation to stress in C. glabrata

• Loss of the deSUMOylating enzyme CgUlp2 leads to reduced SUMO protein levels, impaired growth, stress sensitivity, reduced adherence to epithelial cells, and poor tissue colonization in mice

• While direct interactions between UBC6 and SUMOylation components aren't explicitly described in the search results, these pathways likely function coordinately to regulate protein function and stability

Both ubiquitination and SUMOylation are reversible post-translational modifications that influence protein localization, activity, and degradation. The balance between these modifications may be particularly important during stress conditions, when C. glabrata must rapidly adapt its proteome to survive host immune responses and other challenges .

What are the evolutionary implications of UBC6's non-canonical ubiquitination activity?

The evolutionary significance of UBC6's non-canonical ubiquitination capability can be analyzed from several perspectives:

• UBC6 and its homologs are the only E2 enzymes known to mediate serine/threonine ubiquitination via hydroxyester linkages , suggesting this represents a specialized adaptation

• The highly conserved insertion near the active site, containing S89 and H94, appears to be evolutionarily preserved across UBC6 homologs

• C. glabrata is more closely related to Saccharomyces cerevisiae than to other Candida species , suggesting that UBC6's unique properties may have evolutionary origins in non-pathogenic ancestors

Comparative analysis of UBC6 across the Nakaseomyces clade, which includes both human-associated (commensal) and environmental isolates , could provide insights into how this enzyme's non-canonical activity evolved in relation to adaptation to the human host. The conservation of specific structural features enabling hydroxyester bond formation suggests that this capability confers significant selective advantages in certain ecological niches.

How can molecular dynamics simulations enhance our understanding of UBC6 catalytic mechanisms?

Molecular dynamics simulations offer powerful tools for investigating UBC6's unique catalytic properties:

A comprehensive simulation approach might involve:

• Constructing models of UBC6 bound to ubiquitin in various states (free, thioester-linked)

• Simulating interactions with model substrates containing serine/threonine vs. lysine residues

• Calculating energy barriers for different reaction pathways to understand selectivity

• Predicting the effects of mutations (e.g., H94 variants) on catalytic efficiency

Such simulations would complement experimental approaches and provide atomistic insights into the molecular basis of UBC6's non-canonical activity.

What are the remaining unknowns about UBC6 function and regulation in C. glabrata?

Despite significant advances in understanding UBC6, several important questions remain unanswered:

• Physiological substrates: What are the main physiological targets of UBC6-mediated non-canonical ubiquitination in C. glabrata, and how does their modification influence cellular function?

• Regulatory mechanisms: How is UBC6 expression and activity regulated in response to different environmental conditions and stressors?

• E3 partnerships: Which E3 ubiquitin ligases specifically partner with UBC6 in C. glabrata, and how do these partnerships influence substrate selection?

• Structural dynamics: What conformational changes occur in UBC6 during the catalytic cycle, and how do these changes facilitate the unique reactivity toward hydroxylated amino acids?

• Pathogenic relevance: How does UBC6-mediated non-canonical ubiquitination contribute to C. glabrata virulence, host adaptation, and antifungal resistance?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, molecular genetics, and infection models. The development of specific inhibitors targeting UBC6 could also provide valuable tools for dissecting its functions in C. glabrata pathobiology.

How does C. glabrata UBC6 compare to homologs in other fungal species?

A comparative analysis of UBC6 across fungal species reveals both conservation and specialization:

While the search results don't provide exhaustive information on UBC6 across all fungal species, the evolutionary relationship between C. glabrata and S. cerevisiae suggests their UBC6 homologs likely share similar mechanisms. Comparative functional genomics studies across the Nakaseomyces clade could further illuminate patterns of conservation and divergence linked to phenotypic evolution and adaptation to different ecological niches .

What are the potential applications of UBC6 research for antifungal drug development?

Research on C. glabrata UBC6 may inform novel antifungal strategies:

• Target-based drug discovery: UBC6's unique structural features, particularly the H94 residue critical for non-canonical ubiquitination, could be targeted by small-molecule inhibitors

• Pathway modulation: Disrupting UBC6-dependent protein quality control mechanisms might sensitize C. glabrata to existing antifungals

• Combination therapies: Inhibitors of UBC6 might act synergistically with conventional antifungals by compromising stress adaptation capabilities

The development of UBC6-targeted therapeutics would benefit from:

• High-resolution structural data on UBC6 alone and in complex with ubiquitin

• Identification of critical protein-protein interactions in the UBC6-dependent ubiquitination cascade

• Validation of UBC6's role in stress adaptation and virulence using genetic approaches

• High-throughput screening assays for compounds that specifically inhibit non-canonical ubiquitination

Given C. glabrata's reduced susceptibility to azole antifungals , novel targets like UBC6 may offer valuable alternatives for combating this opportunistic pathogen.

What methodological advances are needed to better understand UBC6 in the context of host-pathogen interactions?

Several methodological advances would enhance research on UBC6's role in host-pathogen interactions:

• In vivo ubiquitination profiling: Development of techniques to identify and quantify hydroxyester ubiquitination events during C. glabrata infection

• Conditional expression systems: Tools to modulate UBC6 activity in a temporal and spatial manner during infection

• Tissue-specific infection models: Systems to evaluate how UBC6 contributes to colonization and invasion of different host tissues

• Interactome mapping: Methods to comprehensively identify UBC6 protein partners, including E3 ligases and substrates, under various infection-relevant conditions

• Single-cell approaches: Techniques to examine cell-to-cell variability in UBC6 expression and activity within heterogeneous C. glabrata populations during infection

These methodological advances would bridge the gap between biochemical understanding of UBC6's mechanism and its functional relevance in the complex environment of the human host. Such approaches could reveal how C. glabrata leverages non-canonical ubiquitination to adapt to diverse host niches and evade immune responses.