Recombinant Debaryomyces hansenii ATP-dependent RNA helicase DBP9 (DBP9), partial

What is Debaryomyces hansenii ATP-dependent RNA helicase DBP9?

Debaryomyces hansenii ATP-dependent RNA helicase DBP9 is a protein belonging to the DEAD-box family of RNA helicases. In D. hansenii, it is encoded by the DBP9 gene (DEHA2F12232g) and functions as an ATP-dependent enzyme (EC 3.6.4.13) that unwinds RNA secondary structures . Based on homology studies with the well-characterized S. cerevisiae DBP9, this protein is likely involved in ribosomal RNA processing and ribosome biogenesis, specifically in the synthesis of 60S ribosomal subunits . The protein contains conserved motifs characteristic of DEAD-box helicases, including the ATP-binding and RNA-binding domains that are essential for its enzymatic activity.

How is DBP9 conserved across fungal species?

DBP9 demonstrates significant conservation across diverse fungal species, indicating its essential role in fundamental cellular processes. The protein has been identified in numerous fungal species including Saccharomyces cerevisiae, Candida albicans, Aspergillus niger, Schizosaccharomyces pombe, and Debaryomyces hansenii . While the level of sequence homology varies between species, the functional domains crucial for ATP binding and RNA helicase activity are highly conserved. This evolutionary conservation suggests that DBP9 plays a critical role in ribosomal biogenesis across the fungal kingdom. Comparative sequence analysis reveals that the core DEAD-box motifs remain largely unchanged, while variation occurs primarily in the N-terminal and C-terminal regions that might contribute to species-specific interactions or regulations.

What expression systems are suitable for producing recombinant DBP9?

Multiple expression systems have been validated for the recombinant production of ATP-dependent RNA helicase DBP9, including bacterial (E. coli), yeast, baculovirus, and mammalian cell systems . Each system offers distinct advantages depending on research objectives:

Selection of an appropriate expression system should be guided by specific research requirements, particularly whether native folding and post-translational modifications are critical for the intended studies .

What purification methods yield high-purity recombinant DBP9?

Standard recombinant protein production protocols can achieve greater than 85% purity for DBP9 as determined by SDS-PAGE analysis . A typical purification workflow includes:

• Affinity chromatography (using His-tag, GST-tag, or other fusion tags)

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing

For analytical-grade preparations (>95% purity), additional steps may include:

• Heparin affinity chromatography (exploiting DBP9's nucleic acid binding properties)

• Hydrophobic interaction chromatography

• Removal of affinity tags using site-specific proteases

The purification strategy should be optimized based on the expression system used and the specific requirements of downstream applications.

How does DBP9 contribute to ribosome biogenesis mechanisms?

Based on studies of S. cerevisiae Dbp9p, the D. hansenii homolog likely plays a critical role in the early stages of 60S ribosomal subunit biogenesis . Dbp9p functions as a nucleolar protein that appears to be involved in the stability of early pre-ribosomal particles. Genetic depletion studies in S. cerevisiae showed that absence of Dbp9p results in:

• Deficit in 60S ribosomal subunits

• Appearance of half-mer polysomes (indicative of ribosome assembly defects)

• Decreased stability of early pre-ribosomal particles

• Reduced steady-state levels of 27S precursors to mature 25S and 5.8S rRNAs

• Decreased synthesis of these rRNA precursors

These findings suggest that DBP9 acts early in the 60S subunit assembly pathway, potentially unwinding complex RNA structures to facilitate proper incorporation of ribosomal proteins or interactions with other assembly factors.

What functional interactions does DBP9 have with other proteins?

In S. cerevisiae, Dbp9p has been shown to functionally interact with Dbp6p, another DEAD-box RNA helicase involved in ribosome biogenesis . This interaction is evidenced by:

• Increased Dbp9p dosage efficiently suppressing certain dbp6 mutant alleles

• Synthetic lethality observed in dbp6/dbp9 double mutants

• Weak direct interaction detected in yeast two-hybrid assays

These findings suggest that DBP9 works cooperatively with other RNA helicases in the complex process of ribosome assembly. While specific interaction partners for D. hansenii DBP9 have not been directly characterized, the high conservation of ribosome assembly pathways suggests similar interactions likely exist. Potential research methods to identify D. hansenii DBP9 interaction partners include:

• Co-immunoprecipitation followed by mass spectrometry

• Proximity-dependent biotin identification (BioID)

• Yeast two-hybrid screening

• Synthetic genetic array analysis

How can researchers design effective ATP-dependent helicase activity assays for DBP9?

Several complementary approaches can be employed to assess the ATP-dependent helicase activity of recombinant DBP9:

When designing these assays, researchers should consider:

• Substrate specificity (RNA sequence and structure preferences)

• Optimal reaction conditions (salt concentration, pH, temperature)

• Potential cofactors or stimulatory proteins

• Controls to distinguish ATP-dependent from ATP-independent activities

What structural analysis approaches are most informative for DBP9 characterization?

Multiple structural biology techniques can provide complementary insights into DBP9 structure-function relationships:

For comprehensive structural characterization, an integrated approach using multiple techniques is recommended to address different aspects of DBP9 structure and dynamics.

How should researchers design experiments to compare DBP9 function across fungal species?

Comparative functional analysis of DBP9 across fungal species requires systematic experimental design:

• Sequence-Based Approach:

  • Multiple sequence alignment to identify conserved and divergent regions

  • Phylogenetic analysis to establish evolutionary relationships

  • Prediction of functional domains and motifs

• Complementation Studies:

  • Express heterologous DBP9 in S. cerevisiae dbp9Δ strains

  • Assess rescue of growth defects and ribosome biogenesis phenotypes

  • Create chimeric proteins to map species-specific functional domains

• Biochemical Comparison:

  • Parallel purification of recombinant DBP9 from multiple species

  • Side-by-side analysis of enzymatic parameters (Km, kcat, substrate preference)

  • Thermal stability and conformational properties

• Interaction Network Mapping:

  • Cross-species protein-protein interaction studies

  • Comparative proteomics of DBP9-associated complexes

  • Network analysis to identify conserved and species-specific interactions

What considerations are important when designing DBP9 knockout or depletion experiments?

When designing genetic manipulation experiments to study DBP9 function, researchers should consider:

• Essential Gene Status:

  • DBP9 is essential in S. cerevisiae, necessitating conditional systems rather than direct knockouts

  • Verify essentiality in D. hansenii before attempting genetic manipulations

• Depletion Strategies:

  • Tetracycline-regulatable promoters for controlled expression

  • Auxin-inducible degron tags for rapid protein depletion

  • Temperature-sensitive alleles for conditional inactivation

• Phenotypic Analysis Framework:

  • Growth rate and viability measurements

  • Polysome profile analysis to assess ribosome biogenesis

  • Northern blotting for rRNA processing intermediates

  • Nucleolar morphology examination via fluorescence microscopy

• Timing Considerations:

  • Primary vs. secondary effects (time-course experiments)

  • Partial vs. complete depletion outcomes

  • Compensatory mechanisms after prolonged depletion

Careful experimental design with appropriate controls is essential for distinguishing direct functions of DBP9 from indirect consequences of disrupting ribosome biogenesis.

How can researchers effectively study DBP9's role in pre-ribosomal particle assembly?

To investigate DBP9's specific role in pre-ribosomal particle assembly, researchers should employ a multi-faceted approach:

• RNA-Protein Interaction Analysis:

  • RNA immunoprecipitation (RIP) to identify bound pre-rRNAs

  • CRAC (crosslinking and analysis of cDNAs) to map binding sites at nucleotide resolution

  • RNA-protein pull-down assays with synthetic rRNA fragments

• Compositional Analysis of Pre-Ribosomes:

  • Affinity purification of tagged DBP9 followed by mass spectrometry

  • Comparison of pre-ribosomal composition before and after DBP9 depletion

  • Gradient fractionation to isolate specific pre-ribosomal particles

• Structural Approaches:

  • Cryo-EM analysis of pre-ribosomal particles with and without DBP9

  • Integrative modeling combining various structural and interaction data

  • Single-particle analysis to capture assembly intermediates

• Functional Reconstitution:

  • In vitro assembly assays with purified components

  • Mapping of ATP-dependent structural rearrangements

  • Reconstitution of minimal RNA remodeling systems

These methodologies allow researchers to dissect the specific molecular functions of DBP9 during the complex process of ribosome assembly.

What are the optimal parameters for assessing RNA substrate specificity of DBP9?

Defining the RNA substrate specificity of DBP9 requires systematic analysis of multiple parameters:

Researchers should develop a substrate library that includes:

• Authentic pre-rRNA sequences from the presumed in vivo targets

• Systematic variations of these sequences to identify critical features

• Structurally characterized RNA elements with defined thermodynamic properties

• Competitor RNAs to assess specificity

A combination of biochemical assays (gel-based unwinding assays, fluorescence-based real-time monitoring) and biophysical measurements (isothermal titration calorimetry, surface plasmon resonance) provides the most comprehensive characterization of substrate specificity.

How might DBP9 function be exploited for antifungal research?

The essential nature of DBP9 in fungal ribosome biogenesis presents potential opportunities for antifungal research:

• Selective Targeting Rationale:

  • DBP9 is essential for fungal viability based on S. cerevisiae studies

  • Structural or functional differences from human homologs could allow selective targeting

  • Involvement in a fundamental process (ribosome biogenesis) makes resistance development less likely

• Drug Development Approaches:

  • High-throughput screening for ATP-competitive inhibitors

  • Structure-based design targeting DBP9-specific pockets

  • Allosteric inhibitors disrupting essential protein-protein interactions

  • RNA-competitive compounds blocking substrate binding

• Methodological Considerations:

  • Develop fungal-specific cell-based assays for DBP9 inhibition

  • Establish correlation between biochemical inhibition and antifungal activity

  • Assess spectrum of activity across pathogenic and non-pathogenic fungi

  • Consider potential for resistance development through compensatory mechanisms

Future studies should focus on validating DBP9 as a drug target by demonstrating specific inhibition in pathogenic fungi.

What are the most promising approaches for studying DBP9 in the context of stress responses?

RNA helicases often play roles in cellular adaptation to environmental stresses. To investigate potential stress-related functions of DBP9:

• Expression Analysis Under Stress Conditions:

  • Transcriptomic and proteomic profiling across various stresses

  • Analysis of potential regulatory elements in DBP9 promoter

  • Protein stability and localization changes during stress

• Genetic Interaction Mapping:

  • Synthetic genetic screens under normal versus stress conditions

  • Identification of condition-specific genetic interactions

  • Epistasis analysis with known stress response factors

• Post-translational Modification Characterization:

  • Phosphoproteomic analysis under different conditions

  • Identification of kinases/phosphatases regulating DBP9

  • Functional consequences of stress-induced modifications

• Ribosome Assembly Dynamics:

  • Alterations in pre-ribosome composition during stress

  • Changes in DBP9 association with pre-ribosomes

  • Impact of stress on ribosome heterogeneity and specialization

These approaches could reveal potential regulatory roles of DBP9 beyond its core function in ribosome biogenesis, particularly in stress adaptation mechanisms relevant to D. hansenii's known osmotolerance.