CytHPPK/DHPS Antibody

What is the molecular structure and biological function of DHPS/HPPK?

DHPS (Deoxyhypusine synthase) catalyzes the NAD-dependent oxidative cleavage of spermidine and the subsequent transfer of the butylamine moiety to the epsilon-amino group of a critical lysine residue in the eukaryotic initiation factor 5A (eIF-5A) precursor protein . This conversion of lysine to deoxyhypusine residue represents the first step in the post-translational modification that creates the unusual amino acid hypusine, which is essential for eIF-5A function .

In plants like Arabidopsis thaliana, DHPS is bifunctional, conjoined with 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). The structure consists of:

• N-terminal HPPK domain (residues 1-160)

• Structured linker region (residues 161-202)

• C-terminal DHPS domain (residues 203-483)

The crystal structure reveals that cytDHPS has a typical TIM-barrel fold with eight-stranded β-barrel surrounded by eight α-helices . The enzyme functions as a dimer, with the dimerization interface occurring via the DHPS domain through helices Dα6, Dη3, Dα7, and Dα8 .

What are the recommended applications and dilutions for CytHPPK/DHPS antibodies?

Based on available data, CytHPPK/DHPS antibodies can be used in multiple applications with specific dilution recommendations:

It is crucial to titrate antibodies in each specific testing system to achieve optimal results, as effectiveness can be sample-dependent .

How does antibody reactivity differ across species for DHPS detection?

The species reactivity profile is an essential consideration when selecting an appropriate antibody:

This species reactivity information should guide experimental design, particularly when working with model organisms. When transitioning between species, validation experiments should be conducted to confirm cross-reactivity.

What strategies can optimize Western blot protocols using CytHPPK/DHPS antibodies?

To optimize Western blot protocols with CytHPPK/DHPS antibodies, consider the following methodological approach:

• Sample preparation considerations:

  • The observed molecular weight of DHPS is 35-40 kDa, while the calculated molecular weight is 41 kDa

  • Use positive controls such as HeLa cell lysates, which have demonstrated consistent detection

• Technical parameters:

  • Begin with a 1:500 dilution and adjust as needed within the 1:200-1:1000 range

  • Storage buffer contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which may influence blocking conditions

  • The antibody is purified via antigen affinity methods, which typically provides good specificity

• Protocol modifications:

  • Considering the 3 isoforms produced by alternative splicing, use appropriate gel percentage to resolve potential close bands

  • When troubleshooting, account for the form (liquid) and storage conditions (-20°C) of the antibody

How can researchers verify antibody specificity for DHPS/HPPK?

Establishing antibody specificity requires a multi-faceted validation approach:

• Positive and negative controls:

  • Use cell lines with known DHPS expression (e.g., HeLa cells for Western blot)

  • Compare with DHPS-knockout or knockdown samples

  • Include appropriate isotype controls (Rabbit IgG for both antibodies)

• Cross-validation methods:

  • Implement immunoprecipitation (IP) followed by Western blot to confirm specificity

  • Consider peptide competition assays using the immunogen

  • For antibodies targeting different epitopes, observe consistent localization patterns

• Epitope mapping considerations:

  • Employ hydrogen-deuterium exchange labeling with 2D NMR for precise epitope identification, similar to methods used for other antibody-antigen interactions

  • This approach can identify protected residues that form the antibody binding site

What is the optimal immunoprecipitation workflow for DHPS studies?

For effective immunoprecipitation of DHPS:

• Sample preparation:

  • Use freshly prepared lysates from appropriate cell types (NIH/3T3 cells have shown successful results)

  • Employ gentle lysis buffers that preserve protein-protein interactions

• Antibody amounts:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Scale proportionally for larger or smaller samples

• Technical considerations:

  • The antigen affinity purification method of the antibody supports effective IP applications

  • Consider pre-clearing lysates to reduce non-specific binding

  • Include appropriate controls to validate specific pull-down

• Detection methods:

  • Western blot analysis of IP samples using the recommended WB dilutions (1:200-1:1000)

  • Consider mass spectrometry for identification of interaction partners

How do cytosolic and mitochondrial forms of HPPK/DHPS differ structurally?

The structural differences between cytosolic and mitochondrial HPPK/DHPS in Arabidopsis thaliana reveal interesting evolutionary adaptations:

• Key amino acid substitutions:

  • Loop D5: Glu346 in cytDHPS (Gln417 in mitDHPS) and Glu349 in cytDHPS (Asp420 in mitDHPS)

  • Loop D6: Gly386 in cytDHPS (Ser457 in mitDHPS) and Ile387 in cytDHPS (Val458 in mitDHPS)

• Structural implications:

  • These variable residues are located either on the enzyme surface or facing away from substrate-binding sites

  • This arrangement explains why cytHPPK/DHPS can complement the function of its mitochondrial counterpart in S. cerevisiae

• Conservation patterns:

  • The DHPS active site and pterin-binding pocket base are almost absolutely conserved across species

  • These conservation patterns suggest functional constraints on active site architecture

How can researchers analyze conformational changes in DHPS during catalysis?

Studying the dynamics of DHPS during catalytic events requires sophisticated methodological approaches:

• Crystallographic approaches:

  • Obtaining structures with bound substrates or substrate analogs at different catalytic stages

  • The crystal structure data for Arabidopsis thaliana DHPS (2.6 Å resolution) provides a foundation for such studies

• Loop dynamics analysis:

  • Critical focus on loops D1, D2, D5, D6, and D7, which are involved in catalysis or active site stabilization

  • Loops D1, D2, D4, and D5 are often disordered in crystal structures, suggesting flexibility relevant to function

• Substrate binding analysis:

  • The DHPS structure contains two key binding pockets: a pterin-binding site at the β-barrel opening and a p-ABA binding site closer to the surface formed by loops D1, D2, and D7

  • Monitoring changes in these binding sites during catalysis is essential for understanding mechanism

• Hydrogen-deuterium exchange methods:

  • Employ H-D exchange labeling with 2D NMR to detect changes in solvent accessibility during catalytic events

  • This technique can reveal which regions become protected or exposed during substrate binding and catalysis

What methodological approaches can map epitopes on DHPS recognized by antibodies?

Understanding the precise antibody binding sites on DHPS requires specialized epitope mapping techniques:

• Hydrogen-deuterium exchange with 2D NMR:

  • Immobilize the antibody on a solid support

  • Form antibody-antigen complex in H₂O and transfer to D₂O

  • After various exchange time periods, dissociate the complex under slow H-exchange conditions

  • Analyze remaining hydrogen labels on individual amide sites using 2D NMR

• Data interpretation:

  • Protected residues (showing slowed H-D exchange by factors up to 340-fold) identify the antibody binding site

  • Analysis must consider both the protected residues and their hydrogen-bond acceptors

  • The technique can identify contiguous, exposed protein surfaces that form the antibody binding site

• Validation approaches:

  • Correlate findings with mutagenesis experiments on residues within the proposed epitope

  • Compare with computational epitope prediction methods

  • Consider 3D structural context of identified residues

How should researchers address inconsistent results when using DHPS antibodies?

When facing inconsistent results with DHPS antibodies, implement a systematic troubleshooting approach:

• Antibody storage and handling:

  • Ensure proper storage at -20°C, where antibodies are stable for one year after shipment

  • Aliquoting is unnecessary for -20°C storage, but avoid repeated freeze-thaw cycles

  • Note that 20 μl sizes contain 0.1% BSA, which may affect certain applications

• Sample preparation factors:

  • Consider that DHPS has 3 isoforms produced by alternative splicing

  • The calculated molecular weight (41 kDa) differs from observed Western blot bands (35-40 kDa)

  • Ensure complete denaturation for Western blot applications

• Technical optimizations:

  • Systematically test different antibody dilutions within the recommended range

  • Modify incubation times, temperatures, and washing conditions

  • For immunofluorescence, optimize fixation methods (paraformaldehyde versus methanol)

• Controls and validation:

  • Include positive control samples (HeLa cells for Western blot, NIH/3T3 cells for IP)

  • Use appropriate negative controls (non-immune rabbit IgG)

  • Consider cross-validation with multiple antibodies targeting different DHPS epitopes

What are the critical parameters for immunofluorescence studies using DHPS antibodies?

For successful immunofluorescence studies with DHPS antibodies:

• Fixation optimization:

  • Test multiple fixation methods (4% paraformaldehyde, methanol, acetone)

  • Optimize permeabilization conditions, particularly important for nuclear and cytoplasmic proteins

• Antibody incubation parameters:

  • Though specific IF dilutions aren't provided, begin with manufacturer recommendations and titrate

  • Consider longer primary antibody incubation times (overnight at 4°C) to improve sensitivity

  • Test both standard and amplification-based detection systems

• Subcellular localization considerations:

  • DHPS is involved in post-translational modification of eIF-5A, suggesting presence in protein synthesis compartments

  • Include appropriate markers for co-localization studies (endoplasmic reticulum, ribosomes)

  • For bifunctional HPPK/DHPS in plants, consider dual localization studies with mitochondrial markers

• Image acquisition and analysis:

  • Use appropriate controls for background subtraction and autofluorescence correction

  • Apply consistent exposure and processing parameters across experimental conditions

  • Consider super-resolution microscopy for detailed subcellular localization studies

How might developments in antibody technology enhance DHPS research?

Emerging antibody technologies offer new opportunities for DHPS research:

• Next-generation recombinant antibodies:

  • Single-chain variable fragments (scFvs) or nanobodies against DHPS could provide enhanced specificity

  • These smaller antibody formats may access epitopes unavailable to conventional antibodies

  • Genetic fusion to reporters or affinity tags could enable novel applications

• Proximity labeling applications:

  • Antibody-mediated targeting of enzymes like APEX2 or TurboID to DHPS

  • This approach could identify transient interaction partners during hypusine formation

  • Could provide insights into the dynamic DHPS interactome during cellular stress

• Intrabodies and targeted degradation:

  • Development of intracellularly expressed antibodies (intrabodies) against DHPS

  • Fusion with degrons to create targeted protein degradation systems

  • Could enable temporal control of DHPS levels without genetic manipulation

What methodological advances could address current limitations in DHPS structural studies?

Several methodological frontiers could advance our understanding of DHPS structure and function:

• Cryo-electron microscopy approaches:

  • Single-particle cryo-EM could reveal conformational heterogeneity not captured in crystal structures

  • May provide insights into the dynamics of substrate binding and catalysis

  • Could potentially capture intermediate states during the reaction cycle

• Time-resolved structural biology:

  • X-ray free-electron laser (XFEL) studies for time-resolved crystallography

  • Could capture transient conformational changes during catalysis

  • Would complement existing crystal structure data of DHPS at 2.6 Å resolution

• Integrative structural biology:

  • Combining multiple structural techniques (X-ray crystallography, NMR, SAXS, cryo-EM)

  • Could provide comprehensive view of DHPS dynamics

  • Computational approaches like molecular dynamics simulations based on these structures

• Advanced epitope mapping:

  • Extending hydrogen-deuterium exchange approaches with mass spectrometry

  • Could provide higher-throughput epitope mapping than NMR-based approaches

  • Would enable more comprehensive understanding of antibody recognition sites