Recombinant Debaryomyces hansenii 60S ribosomal protein L37 (RPL37)

What is Debaryomyces hansenii and why is its RPL37 protein significant for research?

Debaryomyces hansenii is a halotolerant yeast species with remarkable ability to grow in environments with high salt concentrations (up to 1M NaCl or KCl). This organism has been extensively studied to understand mechanisms of salt tolerance in eukaryotic microorganisms . The 60S ribosomal protein L37 (RPL37) is a component of the large ribosomal subunit that participates in protein synthesis. RPL37's significance stems from its potential role in cellular stress responses, including adaptation to high-salt environments. Research suggests that ribosomal proteins like RPL37 may have extraribosomal functions beyond their structural roles in ribosomes, including involvement in translational regulation under stress conditions. Understanding D. hansenii's RPL37 could provide insights into salt-stress response mechanisms.

What expression systems are typically used for recombinant production of yeast ribosomal proteins like RPL37?

Recombinant ribosomal proteins can be expressed using various host systems depending on research requirements. Common expression systems include:

• E. coli bacterial expression: Offers high yield and simplicity but may lack eukaryotic post-translational modifications

• Yeast expression systems: Provide more authentic processing for yeast proteins

• Baculovirus/insect cell systems: Useful for larger or more complex eukaryotic proteins

• Mammalian cell expression: Offers the most complete eukaryotic processing capabilities

The choice depends on experimental needs, with consideration of protein purity requirements (typically ≥85% as determined by SDS-PAGE) . For functional studies of D. hansenii RPL37, a yeast expression system might provide the most biologically relevant product, while bacterial expression might be sufficient for structural studies or antibody production.

How can transcriptomic and proteomic approaches be integrated to study RPL37 regulation in D. hansenii under salt stress?

Integrated multi-omics approaches provide powerful insights into protein regulation under stress conditions. For D. hansenii RPL37 under salt stress, researchers should consider:

• Transcriptomic analysis: RNA-Seq to quantify RPL37 mRNA expression changes under varying salt concentrations (NaCl vs. KCl)

• Proteomic analysis: Mass spectrometry to identify protein abundance changes

• Phosphoproteomic analysis: To detect potential regulatory post-translational modifications

• Ribosome profiling: To examine translational efficiency of RPL37 mRNA under stress

Previous studies with D. hansenii have successfully employed continuous cultivations in controlled lab-scale bioreactors to perform integrated multi-omics comparative analyses under high salt conditions . This approach allows for precise control of environmental conditions while sampling for multiple analytical platforms. Data integration should employ statistical methods that account for different data types and scales, such as weighted correlation network analysis or multi-omics factor analysis.

What role might m6A RNA modification play in regulating RPL37 expression in D. hansenii during salt adaptation?

While there is no direct evidence in the search results specifically about m6A modification of RPL37 in D. hansenii, research on other organisms suggests this could be a significant regulatory mechanism. In human cells, studies have shown that RPL37 mRNA can be modified by m6A, which affects its translation efficiency .

For D. hansenii research, investigating this question would require:

• Performing MeRIP-seq (methylated RNA immunoprecipitation sequencing) to identify m6A modifications on D. hansenii transcripts

• RIP assays to determine if m6A readers (like YTHDC1 homologs in yeast) bind to RPL37 mRNA

• Mutation studies of potential m6A sites in RPL37 mRNA to assess functional consequences

• Comparison of m6A patterns between normal and salt-stress conditions

In human systems, m6A modification on RPL37 has been shown to increase protein synthesis in an m6A-dependent manner through binding of m6A readers like YTHDC1 . If similar mechanisms exist in D. hansenii, they might contribute to rapid adaptation to changing salt conditions by modulating translation efficiency of key ribosomal components.

How can CRISPR-Cas9 genome editing be optimized for studying RPL37 function in D. hansenii?

Optimizing CRISPR-Cas9 for studying RPL37 in D. hansenii requires addressing several technical challenges:

• Delivery system optimization:

  • Electroporation parameters specific for D. hansenii cell wall properties

  • Development of suitable plasmid vectors with appropriate promoters and terminators

• sgRNA design considerations:

  • Target selection accounting for D. hansenii genome characteristics

  • Avoiding potential off-target sites through careful bioinformatic analysis

  • Designing appropriate homology arms for HDR (homology-directed repair)

• Functional modifications to consider:

  • Tagged versions (HA, FLAG, GFP) for localization and interaction studies

  • Point mutations in key functional residues rather than complete knockouts (as RPL37 may be essential)

  • Conditional expression systems (e.g., tetracycline-inducible) for essential genes

• Phenotypic analysis strategies:

  • Growth curve analysis under varying salt conditions

  • Polysome profiling to assess effects on translation

  • Protein synthesis measurement using techniques like puromycin incorporation assays

CRISPR editing efficiency in D. hansenii can be enhanced by temporarily inhibiting non-homologous end joining pathways to favor homology-directed repair for precise editing outcomes.

What are the key considerations for purification of recombinant D. hansenii RPL37 while maintaining its functional properties?

Purification of functional recombinant RPL37 from D. hansenii requires careful attention to several factors:

• Expression system selection:

  • Yeast expression systems may provide more native conformation

  • E. coli systems typically yield higher quantities but may require refolding

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Target purity: ≥85% as determined by SDS-PAGE

• Buffer optimization:

  • Inclusion of reducing agents to maintain cysteine residues in zinc finger motifs

  • Addition of zinc ions may be necessary for structural integrity

  • Ionic strength consideration given D. hansenii's halophilic nature

• Functional validation methods:

  • RNA binding assays to confirm biological activity

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to evaluate stability

• Storage conditions:

  • Flash freezing in small aliquots

  • Addition of stabilizers like glycerol (10-20%)

  • Avoidance of repeated freeze-thaw cycles

The purification protocol must be optimized specifically for D. hansenii RPL37, as general protocols for ribosomal proteins may not account for unique properties related to this halotolerant yeast species.

How can researchers design experiments to study interactions between RPL37 and other components of the D. hansenii ribosome?

Studying RPL37 interactions within the D. hansenii ribosomal complex requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Express tagged RPL37 in D. hansenii

  • Lyse cells under conditions that preserve interactions (e.g., 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 7.5)

  • Immunoprecipitate with anti-tag antibodies

  • Identify interacting partners by mass spectrometry

• Proximity labeling approaches:

  • BioID or TurboID fusion to RPL37 for in vivo labeling of proximal proteins

  • APEX2 fusion for electron microscopy validation

• Crosslinking mass spectrometry (XL-MS):

  • Chemical crosslinking of intact ribosomes

  • Digestion and MS analysis to identify crosslinked peptides

  • Computational modeling of interaction interfaces

• Cryo-electron microscopy:

  • Purification of intact D. hansenii ribosomes

  • Structural determination at near-atomic resolution

  • Comparison with known ribosome structures

• Functional validation:

  • Mutational analysis of key interaction interfaces

  • Ribosome assembly assays

  • Translation efficiency measurements

When designing these experiments, researchers should consider salt concentration effects on interactions, as D. hansenii ribosomes may have evolved specific salt-dependent assembly or interaction characteristics .

What techniques are most effective for studying RPL37 post-translational modifications in D. hansenii under salt stress?

Studying post-translational modifications (PTMs) of RPL37 in D. hansenii under salt stress conditions requires sophisticated analytical approaches:

• Mass spectrometry-based methods:

  • Phosphoproteomics: Titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC) enrichment for phosphopeptides

  • Global PTM profiling: Sequential enrichment strategies for different modification types

  • Targeted MS assays: Parallel reaction monitoring (PRM) for specific modified peptides

  • Quantitative approaches: SILAC or TMT labeling to compare PTM levels under different salt conditions

• Site-specific antibodies:

  • Development of antibodies against predicted modification sites

  • Western blotting to monitor modification dynamics

  • Immunofluorescence to visualize subcellular localization of modified protein

• Genetic approaches:

  • Mutation of potential modification sites (Ser/Thr/Tyr for phosphorylation)

  • Phenotypic analysis of mutants under salt stress

  • Complementation studies with wild-type or modified RPL37

• Time-course experiments:

  • Sampling at multiple timepoints after salt exposure

  • PTM dynamics analysis during adaptation response

  • Correlation with other cellular events

The phosphoproteomic analysis approach has been successfully applied to D. hansenii under salt stress conditions and has implicated specific proteins in the response to high sodium concentrations . This suggests similar approaches would be valuable for characterizing RPL37 modifications.

How should researchers interpret changes in RPL37 expression in the context of global translational reprogramming during salt stress?

Interpreting RPL37 expression changes during salt stress requires contextualizing the data within global translational reprogramming:

• Differential analysis framework:

Previous multi-omics studies of D. hansenii under salt stress have shown that sodium and potassium trigger different responses at both expression and regulation of protein activity levels . Changes in RPL37 should be interpreted within this context, noting whether they align with Na⁺-specific or K⁺-specific cellular adaptations.

What experimental controls are essential when studying the effect of D. hansenii RPL37 on bacterial communities, similar to D. hansenii's effect on bacterial lactase gene diversity?

When studying effects of D. hansenii RPL37 on bacterial communities, rigorous experimental controls are necessary:

• Essential control groups:

  • Untreated control group (healthy baseline)

  • Experimental model control (e.g., antibiotic-treated without D. hansenii)

  • Treatment with whole D. hansenii (positive control)

  • Treatment with D. hansenii lacking RPL37 or with mutated RPL37

  • Treatment with purified RPL37 protein only

• Sampling and analysis controls:

  • Time-matched sampling across all groups

  • DNA extraction controls to account for technical variation

  • PCR/sequencing negative controls to detect contamination

  • Multiple reference genes for normalization of expression data

• Statistical approaches:

  • Appropriate sample size determination (minimum n=6 per group based on previous studies)

  • Non-metric multidimensional scaling (NMDS) for community analysis

  • Diversity indices assessment (Chao1, ACE, Simpson, Shannon)

  • Multiple test correction for taxonomic comparisons

Studies investigating D. hansenii's effects on bacterial communities have shown impacts on specific genera like Cupriavidus, Lysobacter, Citrobacter, Enterobacter, and Pseudomonas . Similar methodologies could be applied when studying RPL37-specific effects, with the addition of controls that isolate RPL37's role from other D. hansenii factors.

How can computational modeling help predict the structural and functional impacts of mutations in D. hansenii RPL37?

Computational modeling provides valuable insights into RPL37 structure-function relationships:

• Structural modeling pipeline:

  • Homology modeling using solved ribosome structures as templates

  • Molecular dynamics simulations under varying salt conditions

  • Protein-RNA docking to predict ribosomal RNA interactions

  • Electrostatic surface analysis to identify salt-responsive regions

• Functional prediction methodologies:

  • Conservation analysis across halotolerant and non-halotolerant yeasts

  • Molecular dynamics simulations of wild-type vs. mutant proteins

  • Free energy calculations to assess stability changes

  • In silico mutagenesis and interaction network analysis

• Key parameters to assess for mutations:

• Integration with experimental validation:

  • In silico predictions should guide targeted mutagenesis experiments

  • Computational results can be validated through thermal stability assays

  • Binding studies can confirm predicted interaction changes

What are promising research directions for understanding potential extraribosomal functions of RPL37 in D. hansenii?

Several promising research directions could reveal extraribosomal functions of RPL37 in D. hansenii:

• Subcellular localization studies:

  • Fluorescent protein tagging to track RPL37 localization under different conditions

  • Cellular fractionation combined with Western blotting

  • Comparison of localization patterns between normal and stress conditions

• Interactome analysis beyond the ribosome:

  • Affinity purification-mass spectrometry under non-ribosome preserving conditions

  • Yeast two-hybrid screening against D. hansenii cDNA library

  • Protein microarray analysis to identify novel binding partners

• Phenotypic analysis of regulated overexpression:

  • Conditional expression systems to induce RPL37 independently of other ribosomal proteins

  • Assessment of cellular phenotypes beyond translation (e.g., stress response, cell cycle)

  • Transcriptome analysis to identify pathways affected by RPL37 overexpression

• Evolutionary analysis across yeasts:

  • Comparative genomics focusing on RPL37 sequence divergence in halotolerant vs. non-halotolerant yeasts

  • Identification of D. hansenii-specific features that might indicate specialized functions

  • Functional complementation studies in other yeast species

Research on RPL37 in human cells has revealed roles beyond ribosome structure, including effects on cell proliferation and migration , suggesting that D. hansenii RPL37 might similarly have functions outside the ribosome, potentially related to halotolerance mechanisms or stress adaptation.

How might findings about D. hansenii RPL37 inform biotechnological applications in high-salt environments?

Research on D. hansenii RPL37 has potential biotechnological applications:

• Engineering salt-tolerant expression systems:

  • Incorporation of D. hansenii RPL37 and its regulatory elements into expression vectors

  • Development of salt-resistant protein production strains

  • Creation of synthetic translational machinery optimized for high-salt conditions

• Bioprocess applications:

  • Enhancement of fermentation processes in high-salt environments

  • Development of salt-tolerant starter cultures for food fermentation

  • Bioremediation applications in saline-contaminated environments

• Biomaterial development:

  • Designing salt-stable enzymes based on RPL37 structural insights

  • Creating biosensors for salt stress using RPL37 regulatory elements

  • Developing immobilization techniques for proteins in high-salt conditions

• Comparative analysis with other halotolerant organisms:

The unique adaptations of D. hansenii to high salt environments, potentially involving RPL37's role in translation under stress conditions, could provide valuable insights for biotechnological applications where salt tolerance is desirable .

What are the potential impacts of climate change and increasing environmental salinity on the evolutionary trajectory of RPL37 in yeasts?

Climate change may influence RPL37 evolution in yeasts as environments become more saline:

• Evolutionary research approaches:

  • Comparative genomics of RPL37 across yeast species with different salt tolerances

  • Experimental evolution studies exposing non-halotolerant yeasts to increasing salinity

  • Analysis of natural yeast populations from habitats with changing salinity

  • Reconstruction of ancestral RPL37 sequences to track evolutionary changes

• Predictive modeling frameworks:

  • Population genetics simulations incorporating climate change parameters

  • Molecular evolution models to predict selection pressures on RPL37

  • Structural biology predictions of adaptation mechanisms

• Monitoring approaches:

  • Establishing baseline RPL37 sequence diversity in current yeast populations

  • Temporal sampling from sites experiencing increasing salinity

  • Functional characterization of emerging RPL37 variants

• Potential experimental validations:

  • CRISPR-mediated replacement of modern RPL37 with ancestral variants

  • Competition experiments between strains with different RPL37 variants

  • Fitness measurements under projected future environmental conditions