Recombinant Chicken Deoxyhypusine hydroxylase (DOHH)

What is chicken DOHH and what is its fundamental role in cellular function?

Chicken DOHH (deoxyhypusine hydroxylase/monooxygenase) is a metalloenzyme that catalyzes the second step in the posttranslational modification of eukaryotic translation initiation factor 5A (eIF5A). Specifically, DOHH hydroxylates the deoxyhypusine intermediate to form hypusine, a modified amino acid found exclusively in eIF5A. This modification is essential for the activation of eIF5A, which plays a critical role in cellular proliferation and protein synthesis regulation in eukaryotes .

The enzymatic reaction occurs after deoxyhypusine synthase (DHS) transfers the aminobutyl moiety of spermidine to a specific lysine residue in eIF5A, forming deoxyhypusine. DOHH then hydroxylates this intermediate to complete hypusine synthesis. This two-step modification pathway is highly conserved across eukaryotic species, suggesting its fundamental importance in cellular function .

How can I effectively express recombinant chicken DOHH in laboratory settings?

For successful expression of recombinant chicken DOHH, mammalian cell expression systems are recommended based on commercial production protocols. The approach involves:

• Vector selection: Use expression vectors containing appropriate promoters for mammalian cell expression and a His-tag for purification purposes.

• Cell line selection: Use mammalian cells such as HEK293 or CHO cells for expression, as these properly manage post-translational modifications.

• Transfection optimization: Determine optimal transfection reagent concentrations and DNA:reagent ratios.

• Expression conditions: Culture at +37°C for 24-72 hours post-transfection, monitoring expression levels at different time points.

• Harvest and lysis: Collect cells and lyse using appropriate buffer systems containing protease inhibitors.

After expression, the protein can be purified using nickel affinity chromatography targeting the His-tag, with final storage in PBS buffer at either +4°C for short-term or -20°C to -80°C for long-term storage .

What are the characteristic structural features of chicken DOHH?

Chicken DOHH (Gallus gallus DOHH, GenBank ID: 427066, UniProt ID: Q5ZIP3) shares the characteristic structural features of DOHH proteins across species. Like human DOHH, chicken DOHH is likely composed of HEAT-repeat motifs arranged in a symmetrical manner, forming a superhelical structure. The protein contains conserved histidine and glutamic acid residues that coordinate iron ions essential for its enzymatic activity .

The protein sequence of chicken DOHH is highly conserved when compared to mammalian counterparts, with particular conservation in the amino acid residues involved in iron coordination and substrate binding. This conservation reflects the fundamental importance of DOHH function across eukaryotic species and suggests similar structural arrangements and enzymatic mechanisms .

What quality control measures should be implemented when working with recombinant chicken DOHH?

When working with recombinant chicken DOHH, implement the following quality control measures:

• Purity assessment: Use SDS-PAGE to verify protein purity, which should be >80% as indicated in commercial preparations .

• Endotoxin testing: Employ the LAL (Limulus Amebocyte Lysate) method to ensure endotoxin levels are below 1.0 EU per μg of protein .

• Functional assays: Evaluate enzymatic activity by measuring the hydroxylation of deoxyhypusine to hypusine in eIF5A substrate, using mass spectrometry or amino acid analysis.

• Stability testing: Assess protein stability under different storage conditions to establish optimal handling protocols.

• Structural integrity: Use circular dichroism (CD) spectroscopy to confirm proper folding of the recombinant protein.

Documentation of batch-to-batch consistency is essential, particularly when using different expression batches for ongoing experiments. For long-term studies, aliquot the protein and store at -80°C to maintain activity and prevent freeze-thaw degradation .

How does chicken DOHH activity compare with DOHH from other species, and what are the implications for cross-species studies?

When conducting cross-species studies:

• Substrate specificity: Examine whether chicken DOHH recognizes and hydroxylates deoxyhypusine-modified eIF5A from other species with similar efficiency.

• Kinetic parameters: Compare Km and Vmax values between chicken DOHH and other species' DOHH using purified recombinant enzymes and synthetic or recombinant substrates.

• Inhibitor sensitivity: Evaluate whether inhibitors of human or yeast DOHH show similar potency against chicken DOHH.

• Structural comparisons: Use homology modeling to identify structural differences that might impact function or inhibitor binding.

The evolutionary conservation of DOHH suggests that findings in the chicken model could be translatable to other species, making it a valuable tool for comparative studies of hypusine modification pathway and inhibitor development .

What are the critical factors affecting the stability and enzymatic activity of recombinant chicken DOHH?

Several critical factors influence the stability and enzymatic activity of recombinant chicken DOHH:

• Iron coordination: DOHH is a metalloenzyme that requires iron for catalytic activity. Ensure adequate iron availability in buffers or consider iron supplementation during expression and purification .

• Redox environment: Maintain appropriate redox conditions to prevent oxidation of crucial cysteine residues that might affect iron coordination or protein folding.

• pH stability: Optimal pH range should be determined experimentally, but typically falls between 7.0-8.0 based on similar enzymes.

• Temperature sensitivity: Store at +4°C for short-term use or -20°C to -80°C for long-term storage to maintain enzymatic activity .

• Buffer composition: PBS buffer is recommended for storage, but activity assays may require specialized buffers containing appropriate cofactors .

The presence of histidine and glutamic acid residues involved in iron coordination is critical for maintaining enzymatic activity. Any mutations or modifications affecting these residues could significantly impact function .

How can I design experiments to elucidate the structure-function relationship of chicken DOHH?

To investigate the structure-function relationship of chicken DOHH, consider the following experimental approaches:

• Site-directed mutagenesis: Target conserved residues involved in iron coordination (histidine and glutamic acid residues) and substrate binding to assess their impact on enzymatic activity .

• Domain swapping experiments: Create chimeric proteins between chicken DOHH and other species' DOHH to identify domains responsible for specific functional properties or substrate recognition.

• Structural analysis: Use X-ray crystallography or cryo-EM to determine the three-dimensional structure of chicken DOHH, ideally in complex with its substrate (deoxyhypusine-modified eIF5A).

• Molecular dynamics simulations: Apply computational approaches to model protein dynamics and substrate binding interactions.

• Enzyme kinetics: Compare kinetic parameters of wild-type and mutant forms of chicken DOHH to correlate structural features with catalytic efficiency.

These approaches should be used in combination to develop a comprehensive understanding of how chicken DOHH structure relates to its function in the hypusine modification pathway .

What methodologies can be employed to study the interaction between chicken DOHH and its substrate eIF5A?

To study the interaction between chicken DOHH and its substrate (deoxyhypusine-modified eIF5A), researchers can employ several complementary methodologies:

• Pull-down assays: Use recombinant His-tagged chicken DOHH to pull down deoxyhypusine-modified eIF5A from cell lysates or in vitro reconstitution systems .

• Surface Plasmon Resonance (SPR): Quantitatively measure binding kinetics and affinity parameters between purified chicken DOHH and eIF5A.

• Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of the binding interaction, providing insights into binding mechanism.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map interaction interfaces by identifying regions with altered solvent accessibility upon complex formation.

• Cross-linking coupled with mass spectrometry: Identify specific residues involved in the interaction by cross-linking the protein complex followed by mass spectrometric analysis.

• Fluorescence-based assays: Develop FRET (Förster Resonance Energy Transfer) assays using fluorescently labeled proteins to monitor binding in real-time.

• Co-crystallization: Attempt to crystallize the DOHH-eIF5A complex to obtain structural information about the binding interface at atomic resolution.

When designing these experiments, it's important to ensure that the eIF5A substrate contains the deoxyhypusine modification, as DOHH specifically recognizes this intermediate form. This may require establishing an in vitro system with DHS to generate the substrate or isolating it from cells treated with DOHH inhibitors .

What is the significance of DOHH inhibition in chicken cells and potential applications in agriculture or disease models?

Inhibition of DOHH in chicken cells has several important implications:

• Cell proliferation control: Since hypusine modification is essential for eIF5A function and cellular proliferation, inhibiting DOHH could potentially regulate abnormal cell growth in avian disease models .

• Viral replication interference: eIF5A has been implicated in viral replication processes. DOHH inhibition could potentially interfere with the replication of avian viruses, as suggested by studies with recombinant Newcastle Disease Virus (NDV) .

• Developmental biology: DOHH inhibition could serve as a tool to study the role of hypusine-modified eIF5A in chicken embryonic development and differentiation.

• Agricultural applications: Understanding DOHH function could lead to novel approaches for improving poultry health, potentially through targeted interventions in the hypusine pathway.

• Comparative biology: Chicken DOHH inhibition studies can provide insights into evolutionary conservation of hypusine pathway functions across species, given that DOHH is lethal when suppressed in higher multicellular eukaryotes like C. elegans and Drosophila but not in S. cerevisiae .

Experimental approaches might include:

• Small molecule inhibitors developed against human DOHH

• RNA interference or CRISPR-mediated knockdown of DOHH in chicken cell lines

• Development of chicken-specific DOHH inhibitors based on structural information

The findings from such studies could have significant implications for avian biotechnology, disease control, and comparative cell biology research .

What are the optimal conditions for enzymatic assays using recombinant chicken DOHH?

For optimal enzymatic assays using recombinant chicken DOHH, the following conditions should be considered:

• Substrate preparation: Use recombinant eIF5A containing the deoxyhypusine modification, which can be generated by treating eIF5A with DHS in the presence of spermidine and NAD+ .

• Buffer composition:

  • 50 mM Tris-HCl, pH 7.5-8.0

  • 1-2 mM DTT (to maintain reducing conditions)

  • 50-100 μM ferrous ammonium sulfate (as iron source)

  • 1-5 mM ascorbic acid (to maintain iron in reduced state)

  • 50-100 μM of substrate (deoxyhypusine-modified eIF5A)

• Assay temperature: Typically 37°C to match physiological conditions.

• Reaction time: 30-60 minutes, with aliquots taken at different time points to establish reaction kinetics.

• Detection methods:

  • HPLC analysis of hydrolyzed protein to detect hypusine formation

  • Mass spectrometry to detect the mass shift associated with hydroxylation

  • Radiolabeled substrate approach using [3H]-labeled spermidine in the DHS reaction, followed by measurement of incorporated radioactivity

• Controls:

  • Negative control: reaction mixture without DOHH enzyme

  • Positive control: known active DOHH preparation (e.g., human DOHH)

  • Inhibition control: reaction in the presence of known DOHH inhibitors like metal chelators

Ensure that recombinant chicken DOHH is stored in PBS buffer at +4°C for short-term use or -20°C to -80°C for long-term storage to maintain enzymatic activity .

How can challenges in the expression and purification of enzymatically active chicken DOHH be addressed?

Researchers face several challenges when expressing and purifying enzymatically active chicken DOHH. Here are methodological approaches to address these issues:

• Low expression levels:

  • Optimize codon usage for the expression system

  • Evaluate different promoters (CMV, EF1α) for mammalian expression

  • Consider inducible expression systems to reduce potential toxicity

  • Test different cell lines (HEK293, CHO, insect cells) for optimal expression

• Protein solubility:

  • Express at lower temperatures (28-30°C) to promote proper folding

  • Include solubility-enhancing tags (e.g., GST, MBP) in addition to His-tag

  • Optimize lysis buffer composition (add glycerol, mild detergents)

  • Consider co-expression with molecular chaperones

• Metalloenzyme-specific issues:

  • Supplement growth media and purification buffers with iron source

  • Avoid metal chelators (EDTA) during purification

  • Include reducing agents to maintain iron in the appropriate oxidation state

• Purification strategy:

  • Implement a two-step purification process:
    a. Affinity chromatography using His-tag or GST-tag
    b. Size exclusion chromatography for higher purity

  • Consider on-column refolding protocols if inclusion bodies form

• Activity preservation:

  • Add stabilizing agents (glycerol 10-20%) to final storage buffer

  • Store in small aliquots to avoid freeze-thaw cycles

  • Consider lyophilization as alternative storage method

Following these strategies should yield recombinant chicken DOHH with purity >80% and endotoxin levels <1.0 EU per μg protein, suitable for research applications .

How can recombinant chicken DOHH be utilized in comparative studies of the hypusine modification pathway across species?

Recombinant chicken DOHH serves as a valuable tool for comparative studies of the hypusine modification pathway across species, offering insights into evolutionary conservation and species-specific variations:

• Cross-species substrate utilization:

  • Test whether chicken DOHH can hydroxylate deoxyhypusine-modified eIF5A from different species (human, mouse, yeast)

  • Compare enzymatic efficiency (kcat/Km) across substrates from different species

  • Identify species-specific structural determinants of substrate recognition

• Evolutionary analysis:

  • Create phylogenetic trees based on DOHH sequences to trace evolutionary relationships

  • Compare with eIF5A and DHS phylogenies to identify co-evolutionary patterns

  • Correlate sequence conservation with functional importance of specific residues

• Functional complementation studies:

  • Determine if chicken DOHH can rescue growth defects in DOHH-deficient yeast or mammalian cells

  • Assess the importance of species-specific domains through chimeric proteins

  • Compare the necessity of hypusine modification across species (e.g., essential in multicellular organisms but not in S. cerevisiae)

• Inhibitor sensitivity profiling:

  • Compare the sensitivity of chicken DOHH to known inhibitors of human DOHH

  • Identify species-specific differences in inhibitor binding sites

  • Develop species-selective inhibitors based on structural differences

• Cellular localization and regulation:

  • Compare subcellular localization patterns of DOHH across species

  • Identify species-specific regulatory mechanisms controlling DOHH expression and activity

These comparative approaches can reveal fundamental aspects of eIF5A activation that have been conserved through evolution while also highlighting adaptations specific to avian or other lineages .

How might inhibitors of chicken DOHH be developed as research tools or therapeutic agents?

Development of chicken DOHH inhibitors represents an important research direction with potential applications in both basic science and applied fields:

• Rational inhibitor design approaches:

  • Structure-based design using homology models of chicken DOHH based on human DOHH structure

  • Metal chelator-based inhibitors targeting the iron center essential for DOHH activity

  • Substrate competitive inhibitors that mimic deoxyhypusine-modified eIF5A

  • Allosteric inhibitors targeting non-catalytic regulatory sites

• Screening methodologies:

  • High-throughput screening of chemical libraries using purified recombinant chicken DOHH

  • Fragment-based screening to identify building blocks for inhibitor development

  • Repurposing of known human DOHH inhibitors with modifications to enhance specificity for chicken DOHH

• Validation assays:

  • In vitro enzymatic assays measuring inhibition of hypusine formation

  • Cell-based assays in chicken cell lines measuring proliferation inhibition

  • Target engagement assays to confirm binding to DOHH in cellular context

  • Selectivity profiling against human DOHH and other related enzymes

• Potential applications as research tools:

  • Temporal control of DOHH activity in developmental studies

  • Investigation of hypusine-dependent versus independent functions of eIF5A

  • Studies of tissue-specific requirements for DOHH activity in avian systems

• Therapeutic applications:

  • Control of avian viral infections that depend on hypusine-modified eIF5A

  • Potential applications in avian cancer models

  • Regulation of inflammatory responses in avian disease models

The development of such inhibitors would be facilitated by detailed structural information on chicken DOHH and would provide valuable tools for studying the hypusine modification pathway in avian systems .

What is currently known about the regulatory mechanisms controlling chicken DOHH expression and activity?

The regulatory mechanisms controlling chicken DOHH expression and activity remain largely unexplored, but some insights can be inferred from studies of the hypusine pathway in other species and general principles of enzyme regulation:

• Transcriptional regulation:

  • Promoter analysis of the chicken DOHH gene (Gene ID: 427066) could reveal tissue-specific regulatory elements

  • Transcription factors involved in proliferation and development likely regulate DOHH expression

  • Cell cycle-dependent expression patterns may exist, given the role of hypusine modification in proliferation

• Post-transcriptional control:

  • Alternative splicing may generate DOHH variants with different activities

  • mRNA stability and translation efficiency could be regulated in response to cellular conditions

  • microRNA targeting of DOHH transcripts may provide additional regulatory control

• Post-translational regulation:

  • Phosphorylation or other modifications could modulate DOHH activity

  • Protein-protein interactions might regulate localization or substrate access

  • Cellular iron availability likely impacts DOHH activity given its nature as a metalloenzyme

• Metabolic regulation:

  • Connection to polyamine metabolism through the use of spermidine in the first step of the hypusine pathway

  • Coordination with eIF5A and DHS expression levels to maintain stoichiometric balance

  • Potential oxygen-dependent regulation of hydroxylase activity

• Experimental approaches to study regulation:

  • Promoter reporter assays to identify regulatory elements

  • Pulse-chase experiments to determine protein half-life

  • Proximity labeling to identify interaction partners

  • Metabolic profiling to understand connections to other pathways

Understanding these regulatory mechanisms would provide insights into how the hypusine modification pathway is integrated with other cellular processes and how it responds to developmental cues and stress conditions in avian systems .