NUDT14 Antibody

What is NUDT14 and why is it important to study?

NUDT14 is a UDP-glucose pyrophosphatase (EC 3.6.1.45) that hydrolyzes UDP-glucose (UDPG) to produce glucose 1-phosphate and UMP . As a member of the NUDIX hydrolase family, it plays a significant role in controlling nucleotide sugar metabolism. UDPG serves as the sugar donor in numerous glycosylation reactions, including those involved in glycogen synthesis . Beyond its role in glucose metabolism, recent research has revealed that NUDT14 can also hydrolyze ADP-ribose and ADP-glucose .

Studying NUDT14 is particularly important because it is frequently expressed at elevated levels in cancer cells . This observation, combined with its enzymatic activity, suggests potential roles in cancer metabolism and progression that warrant further investigation. The protein's challenging characteristics—particularly its smaller and more exposed active site—have made it a difficult target to study, which has limited our understanding of its complete functional profile .

What types of NUDT14 antibodies are available for research applications?

Current commercially available NUDT14 antibodies include several variants targeting different epitopes of the protein:

• N-terminal specific polyclonal antibodies (such as those targeting amino acids 1-100)

• Full-length protein antibodies (covering amino acids 1-222)

• Middle region antibodies (amino acids 151-200)

Most available antibodies are rabbit polyclonal preparations that have been affinity-purified . The immunogens typically used are synthetic peptides directed toward specific regions of human NUDT14 . While polyclonal antibodies dominate the market, they vary in their specific epitope targeting, which has implications for experimental applications.

What applications are NUDT14 antibodies validated for?

NUDT14 antibodies have been validated for several research applications:

Researchers should note that not all antibodies are validated for all applications. When selecting an antibody, confirm that it has been specifically validated for your intended experimental procedure .

What species reactivity can be expected from NUDT14 antibodies?

NUDT14 antibodies show varying degrees of cross-reactivity across species due to sequence homology. Based on available data, the following reactivity profiles can be expected:

• Human: 100% reactivity (most antibodies are designed against human sequences)

• Cow: 100% reactivity for some antibodies

• Mouse: Approximately 79-82% reactivity (variable between antibodies)

• Rat: Approximately 85-86% reactivity

• Pig: Approximately 93% reactivity

When planning cross-species experiments, it's advisable to select antibodies with validated reactivity for your species of interest. The reactivity percentages reflect sequence identity in the epitope regions rather than absolute detection efficiency, so validation in your specific experimental system remains essential .

How should researchers design experiments to distinguish between NUDT14 and related NUDIX family members?

The NUDIX hydrolase family contains several members with similar substrate profiles, making specific detection critical. When designing experiments to distinguish NUDT14 from other NUDIX proteins (particularly NUDT5):

• Antibody selection considerations: Choose antibodies targeting unique epitopes not conserved across the NUDIX family. The N-terminal domain is particularly useful as it has now been resolved and shows distinct structural features .

• Functional validation approach: Recent research has identified compound 9 as a potent dual NUDT5 and NUDT14 inhibitor . To distinguish between these proteins:

  • Use selective inhibitors in parallel experiments

  • Compare results with TH5427, which shows exquisite selectivity for NUDT5 over NUDT14

  • Employ complementary genetic approaches (siRNA, CRISPR) to validate antibody findings

• Structural analysis: The resolved cocrystal structure of NUDT14 reveals that while some active site residues are conserved between NUDT5 and NUDT14 (Y17/Y36 and W34/W46), there are notable differences :

  • NUDT14 inhibitors interact with L107 rather than the R51 found in NUDT5

  • The N-terminal domain of NUDT14 consists of β-sheets intertwined with the NUDIX domain of the second subunit

These structural differences can guide experimental design and interpretation when studying NUDT14 specifically.

What methodological approaches can be used to study NUDT14 catalytic activity?

Several complementary methodologies can be employed to study NUDT14 enzymatic function:

• In vitro enzymatic assays:

  • Monitor UDPG hydrolysis to glucose 1-phosphate and UMP using HPLC or coupled enzyme assays

  • Measure ADP-ribose and ADP-glucose hydrolysis as alternative substrates

  • Determine enzyme kinetics (Km, Vmax, kcat) under various conditions to understand catalytic properties

• Inhibitor-based approaches:

  • Use BTK inhibitor ibrutinib (compound 1) which demonstrates dual inhibition of NUDT5 and NUDT14 (IC50 = 0.990 ± 0.110 μM for NUDT14)

  • Employ compound 9, a more selective and potent dual NUDT5/NUDT14 inhibitor with minimal BTK activity

  • Determine IC50 values using dose-response curves in enzymatic assays

• Cellular target engagement assays:

  • NanoBRET for NUDT5 target engagement assessment

  • HiBiT CETSA (Cellular Thermal Shift Assay) for NUDT14, which can detect thermal stabilization (ΔTm = 5.5 ± 0.3 for compound 9)

  • Surface Plasmon Resonance (SPR) screening to assess binding specificity across the NUDIX family

These methodologies provide complementary information about NUDT14 catalytic activity, inhibitor potency, and cellular relevance.

What are the key structural insights about NUDT14 that influence antibody selection and experimental design?

Recent structural biology advances have revealed critical features of NUDT14 that should inform experimental design:

• N-terminal domain structure: The previously unresolved N-terminal domain consists of β-sheets intertwined with the NUDIX domain of the second subunit . This unique structural arrangement suggests:

  • Antibodies targeting this region may recognize conformational epitopes dependent on dimerization

  • Native conditions may be required for certain antibody applications

• Active site architecture:

  • Key residues in the NUDT14 active site include Y17 and W34, which form π-π stacking interactions with ligands

  • D35 forms hydrogen bonds with inhibitors

  • L107 engages in hydrophobic interactions with aromatic substituents of inhibitors

• Comparison with NUDT5:

  • Despite some conserved residues (Y17/Y36 and W34/W46), inhibitors interact differently with the two proteins

  • R51, important for ligand binding in NUDT5, is not conserved in NUDT14

  • These differences explain the selectivity of compounds like TH5427 for NUDT5 over NUDT14

Understanding these structural features can guide antibody epitope selection and help interpret experimental results when studying NUDT14 function.

How can researchers optimize NUDT14 antibody-based detection in cancer research applications?

NUDT14 is frequently expressed at elevated levels in cancer cells , making it potentially relevant for cancer research. To optimize antibody-based detection in this context:

• Sample preparation optimization:

  • Compare multiple lysis protocols to preserve epitope integrity

  • Evaluate the need for phosphatase or protease inhibitors to prevent post-lysis modifications

  • Consider nuclear extraction protocols, as NUDIX hydrolases may have nuclear functions

• Signal amplification strategies:

  • For tissues with lower expression, consider biotin-conjugated secondary antibodies with streptavidin-HRP

  • Use tyramide signal amplification for immunohistochemistry applications

  • Implement multiplex immunofluorescence to correlate NUDT14 expression with other cancer markers

• Control and validation approaches:

  • Include positive controls from cell lines with known NUDT14 overexpression

  • Implement siRNA knockdown controls to validate signal specificity

  • Consider dual-antibody approaches using antibodies targeting different epitopes to confirm specificity

• Quantification methods:

  • Develop standardized scoring systems for immunohistochemistry applications

  • Use digital image analysis for consistent quantification

  • Correlate protein levels with mRNA expression data when possible

These approaches can enhance the reliability and sensitivity of NUDT14 detection in cancer research applications.

What are common technical challenges when using NUDT14 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with NUDT14 antibodies:

• Specificity concerns:

  • Challenge: Cross-reactivity with other NUDIX family members

  • Solution: Validate antibody specificity using knockout/knockdown controls and parallel detection with multiple antibodies targeting different epitopes

• Sensitivity limitations:

  • Challenge: Low endogenous expression in certain cell types

  • Solution: Optimize sample preparation, consider using concentrated lysates, implement signal amplification methods, or use more sensitive detection systems

• Background signal:

  • Challenge: Non-specific binding, particularly with polyclonal antibodies

  • Solution: Optimize blocking conditions (test BSA vs. non-fat milk), adjust antibody concentration, extend washing steps, and consider pre-adsorption of antibodies

• Epitope masking:

  • Challenge: Protein-protein interactions or post-translational modifications may mask epitopes

  • Solution: Test multiple antibodies targeting different regions of NUDT14, optimize lysis conditions to disrupt protein complexes

A systematic approach to troubleshooting these issues involves testing multiple variables in parallel and maintaining detailed records of experimental conditions.

How can researchers study the association between NUDT14 and potential interacting partners?

Investigating NUDT14 protein-protein interactions requires specialized approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Forward approach: Immunoprecipitate NUDT14 using validated antibodies and identify binding partners by mass spectrometry or immunoblotting

  • Reverse approach: Immunoprecipitate suspected interacting partners and probe for NUDT14

  • Cross-linking prior to lysis can capture transient interactions

• Proximity-based methods:

  • BioID or TurboID approach: Fuse NUDT14 to a promiscuous biotin ligase to biotinylate proteins in close proximity

  • APEX2 proximity labeling: Similar principle using peroxidase-catalyzed biotinylation

  • These methods can identify both stable and transient interactions

• Microscopy-based techniques:

  • Proximity Ligation Assay (PLA): Detects proteins within 40 nm of each other in fixed samples

  • FRET or BRET: For studying interactions in living cells

  • Co-localization analysis using super-resolution microscopy

• Functional validation:

  • Mutational analysis of interaction domains

  • Competition assays with peptides or small molecules

  • Functional readouts (enzymatic activity, cellular localization) upon disruption of interactions

When designing these experiments, consider the potential for NUDT14 interactions to be substrate-dependent or regulated by cellular conditions such as stress or metabolic state.

What approaches can be used to correlate NUDT14 expression with functional outcomes in cell-based systems?

To establish functional significance of NUDT14 expression:

• Genetic manipulation strategies:

  • CRISPR-Cas9 knockout or knockdown via siRNA/shRNA

  • Overexpression systems using tagged constructs

  • Inducible expression systems to study dose-dependent effects

• Pharmacological inhibition:

  • Use compound 9 as a dual NUDT5/NUDT14 inhibitor

  • Compare with selective inhibitors of related pathways

  • Implement dose-response and time-course studies

• Metabolic assessments:

  • Measure UDP-glucose levels and related metabolites

  • Assess glycogen synthesis rates

  • Monitor ADP-ribose pools after DNA damage

• Functional readouts based on cellular context:

  • For cancer cells: Proliferation, migration, invasion assays

  • Stress response: Sensitivity to DNA-damaging agents

  • Metabolism: Glycolytic rates, energy charge, nucleotide pools

When interpreting results, consider that NUDT14 inhibition may not show phenotypes in all cellular contexts due to functional redundancy with other NUDIX hydrolases, particularly NUDT5 .

What unresolved questions about NUDT14 represent opportunities for novel antibody-based investigations?

Several knowledge gaps present opportunities for researchers:

• Tissue-specific expression patterns:

  • Current understanding of NUDT14 expression across normal tissues remains limited

  • Opportunity: Develop high-throughput immunohistochemistry panels to map expression across tissue types and developmental stages

• Post-translational modifications:

  • Little is known about how NUDT14 activity is regulated

  • Opportunity: Develop modification-specific antibodies (phospho, acetyl, ubiquitin) to study regulatory mechanisms

• Subcellular localization dynamics:

  • Nuclear versus cytoplasmic functions remain to be elucidated

  • Opportunity: Use super-resolution microscopy with subcellular markers to map precise localization under various conditions

• Cancer-specific alterations:

  • While NUDT14 is overexpressed in some cancers, the functional consequences are unclear

  • Opportunity: Correlate expression with patient outcomes and therapy response using tissue microarrays

• Interaction with metabolic pathways:

  • NUDT14's role in glucose metabolism suggests broader metabolic impacts

  • Opportunity: Combine antibody-based detection with metabolomic approaches

These research directions could significantly advance our understanding of NUDT14 biology and its potential as a therapeutic target.

How might NUDT14 antibodies be utilized in translational research applications?

The potential translational applications of NUDT14 antibodies include:

• Biomarker development:

  • Evaluate NUDT14 as a diagnostic or prognostic marker in cancer types where expression is elevated

  • Develop standardized immunohistochemistry protocols for clinical laboratory implementation

  • Correlate expression patterns with clinical outcomes and treatment responses

• Therapeutic target validation:

  • Use antibody-based detection to confirm target engagement of NUDT14-directed small molecule inhibitors

  • Evaluate expressional changes in response to other therapeutic interventions

  • Identify patient subgroups most likely to benefit from NUDT14-targeted therapies

• Combination therapy rationales:

  • Investigate how NUDT14 expression or activity modifies response to standard therapies

  • Use antibody detection to develop pharmacodynamic markers for clinical trials

  • Identify synthetic lethal interactions that could inform combination approaches

• Drug development support:

  • Employ antibodies in target engagement assays like cellular thermal shift assays (CETSA)

  • Develop cell-based screening systems using antibody-based readouts

  • Monitor on-target versus off-target effects of candidate compounds

These applications highlight how NUDT14 antibodies can bridge fundamental research findings to clinical applications, particularly in oncology where NUDT14 expression appears elevated in certain contexts .