NUDT14 Human

What is the primary enzymatic function of NUDT14 and how can it be measured?

NUDT14, also known as uridine diphosphate glucose pyrophosphatase, primarily functions to hydrolyze ADP-ribose into ribose 5-phosphate and AMP, and UDP-glucose to glucose 1-phosphate and UMP. This 222 amino acid cytoplasmic protein contains one nudix hydrolase domain and exists as a homodimer in its functional state . To measure enzymatic activity, researchers typically employ spectrophotometric assays to monitor substrate conversion, HPLC to separate and quantify reaction products, or coupled enzyme assays that link NUDT14 activity to detectable signals.

When designing these assays, it's critical to include magnesium as a cofactor since NUDT14 requires this metal ion for catalytic activity. Optimal buffer conditions for measuring NUDT14 activity typically include 20mM Tris-HCl (pH 8.0) with 0.1M NaCl, 10% glycerol, and 1mM DTT, which mimics the physiological environment in which the enzyme functions .

What expression systems are most effective for producing recombinant NUDT14?

Recombinant human NUDT14 is most commonly expressed in E. coli systems, which provide high yield and relatively simple purification protocols. The methodology involves:

• Cloning the NUDT14 gene into an appropriate expression vector

• Transformation into an E. coli expression strain

• Induction of protein expression

• Cell lysis followed by purification using chromatography techniques

For affinity purification, NUDT14 is often expressed with a polyhistidine tag at the N-terminus, allowing purification by immobilized metal affinity chromatography (IMAC) . The resulting recombinant protein typically has a molecular mass of approximately 26.5 kDa (for a 245 amino acid construct including the His-tag), which can be confirmed by MALDI-TOF mass spectrometry. Final purity of >95% can be achieved and verified by SDS-PAGE analysis .

How should NUDT14 protein be stored to maintain activity for experimental use?

Based on standard protocols for recombinant NUDT14, optimal storage conditions include:

• Short-term storage (1-2 weeks): 4°C in buffer containing 20mM Tris-HCl (pH 8.0), 0.1M NaCl, 10% glycerol, and 1mM DTT

• Long-term storage: Aliquot and store at -20°C or -70°C to prevent freeze-thaw cycles

The inclusion of 10% glycerol serves as a cryoprotectant, while DTT (1mM) helps maintain protein stability by preventing oxidation of cysteine residues. When thawing stored protein, gradual temperature transition is recommended to prevent protein denaturation. After thawing, protein activity should be verified using appropriate enzymatic assays before experimental use .

What are the structural characteristics of NUDT14 and how do they relate to its function?

NUDT14 is a 222 amino acid cytoplasmic protein containing one nudix hydrolase domain. Key structural features include:

• A homodimeric quaternary structure essential for catalytic activity

• The N-terminal domain consists of β-sheets that intertwine with the NUDIX domain of the second subunit

• An active site containing key residues involved in substrate binding and catalysis

• Binding sites for magnesium cofactors

Crystal structure studies, particularly those involving inhibitor-bound NUDT14, have revealed important details about the active site architecture. The active site includes residues W34 of chain A and Y17 of chain B, which form π–π stacking interactions with heterocyclic compounds. Additional residues like D35 form hydrogen bonds with substrates, while L107 participates in hydrophobic interactions . Understanding these structural elements is crucial for designing enzyme inhibitors and studying substrate specificity.

What methodologies are most effective for studying NUDT14-protein interactions in cellular contexts?

To investigate NUDT14-protein interactions in cellular contexts, several complementary methodologies have proven effective:

• Two-hybrid screening - Can identify potential protein interaction partners in a cellular environment. This has been successfully used to detect the interaction between NUDT14 and viral proteins like HCMV RL13 .

• Co-immunoprecipitation (Co-IP) - Validates protein-protein interactions in human cell lines (e.g., HEK293). This technique has confirmed specific interactions between NUDT14 and binding partners identified through other methods .

• GST pull-down assays - Provides in vitro confirmation of direct protein interactions, as demonstrated in studies of NUDT14 interaction with viral proteins .

• Fluorescence confocal microscopy - Enables visualization of co-localization between NUDT14 and interacting proteins. This technique has shown NUDT14 co-localization with the HCMV RL13 protein in the cell membrane and cytoplasm .

• Cellular thermal shift assay (CETSA) - Particularly the HiBiT CETSA has been established for NUDT14 to evaluate target engagement of small molecules in living cells. This method can detect thermal stabilization of NUDT14 upon binding of inhibitors .

These methodologies collectively provide robust evidence for specific protein-protein interactions and can elucidate the biological consequences of these interactions.

How does NUDT14 expression modulation affect viral replication, and what techniques best measure this effect?

Research has demonstrated that NUDT14 expression levels can impact viral replication, particularly for human cytomegalovirus (HCMV). To study this relationship:

• RNA interference (RNAi) - NUDT14-specific small interfering RNAs (siRNAs) can reduce expression levels. Studies have shown that decreasing NUDT14 expression via siRNAs increases viral DNA copies in HCMV-infected cells .

• Stable overexpression systems - Cell lines stably expressing NUDT14 can be established to study the effect of increased NUDT14 levels. Interestingly, research has shown that while knockdown increases viral DNA levels, overexpression does not significantly affect viral DNA levels in HCMV infected cells .

• Quantitative PCR (qPCR) - This technique precisely measures viral DNA copy numbers in infected cells under different NUDT14 expression conditions.

• Plaque assays - Can determine infectious viral titers to complement qPCR data.

When designing these experiments, it's important to include appropriate controls and to verify that the observed effects are specific to NUDT14 modulation rather than off-target effects. The asymmetric effect of NUDT14 knockdown versus overexpression suggests complex regulatory mechanisms that warrant further investigation.

What approaches are available for developing and characterizing NUDT14 inhibitors?

Development of NUDT14 inhibitors is an emerging area with recent breakthroughs. The methodological approach includes:

• Structure-based design - Utilizing crystal structures of NUDT14 to design compounds that bind to the active site. A recent breakthrough yielded the first inhibitor-bound structure of NUDT14 at 1.82 Å resolution .

• Surface Plasmon Resonance (SPR) - This technique directly measures binding affinity between NUDT14 and potential inhibitors. For example, compound "9" demonstrated potent direct binding to human NUDT14 with a KD of approximately 400 nM .

• Crystallography - Co-crystallization trials with candidate inhibitors provide detailed structural information. Recent studies revealed that inhibitors can occupy the active site of NUDT14 where heterocyclic cores engage in π–π stacking interactions with specific residues (W34 of chain A and Y17 of chain B) .

• Cell-based target engagement assays - The HiBiT CETSA (Cellular Thermal Shift Assay) has been established specifically for NUDT14 to confirm inhibitor binding in living cells .

The development of selective NUDT14 inhibitors provides valuable chemical probes for studying NUDT14 biological function and potentially therapeutic applications.

What are the methodological challenges in studying the relationship between NUDT14 and ADP-ribosylation dynamics?

• Redundancy in ADP-ribose processing - Experiments with NUDT14 inhibitors in human osteosarcoma U2OS ARH3 KO cells did not significantly affect protein-bound ADP-ribose levels, suggesting redundant or alternative mechanisms . This complicates the isolation of NUDT14-specific effects.

• Transient nature of ADP-ribosylation - ADP-ribosylation is often transient, requiring special techniques for visualization:

  • Use of poly(ADP-ribose) glycohydrolase (PARG) inhibitors to stabilize poly(ADP-ribose) chains

  • Development of specialized antibodies to detect mono- and poly-ADP-ribosylation

  • Mass spectrometry approaches to identify ADP-ribosylation sites

• Distinguishing free vs. protein-bound ADP-ribose metabolism - While NUDT14 acts on free ADP-ribose, connecting this activity to protein-bound ADP-ribosylation requires specialized metabolic tracing experiments and correlation analyses between free ADP-ribose levels and protein ADP-ribosylation.

• Establishing biological significance - Determining the physiological relevance of NUDT14's ADP-ribose hydrolyzing activity requires phenotypic analysis of NUDT14 knockout systems and integration with broader NAD+ metabolism networks.

These challenges highlight the need for complementary approaches and careful experimental design when investigating NUDT14's role in ADP-ribosylation dynamics.

How can researchers effectively distinguish between the direct and indirect effects of NUDT14 inhibition in cellular systems?

Differentiating direct from indirect effects of NUDT14 inhibition requires carefully designed experimental approaches:

• Dose-response and time-course analyses:

  • Direct effects typically manifest at lower inhibitor concentrations and earlier time points

  • Concentration-dependent effects should correlate with the known binding affinity of the inhibitor

• Orthogonal validation approaches:

  • Compare phenotypes from pharmacological inhibition with genetic knockdown/knockout

  • Use structurally distinct inhibitors targeting the same site

  • Employ rescue experiments with inhibitor-resistant NUDT14 mutants

• Target engagement confirmation:

  • Cellular thermal shift assay (CETSA) to verify binding in intact cells

  • NanoBRET assays to measure target engagement in live cells, which has been validated for NUDT14 inhibitors

  • In-cell activity assays measuring NUDT14 substrate accumulation

• Control experiments with related enzymes:

  • Test effects in cells lacking NUDT14 expression

  • Assess inhibitor specificity against related NUDIX hydrolases

  • Use of known selective inhibitors of related pathways as comparators

The recent development of the HiBiT CETSA specifically for NUDT14 provides a valuable tool for confirming on-target engagement in cellular systems, helping researchers distinguish specific effects from off-target activities .

How do NUDT14 and NUDT5 differ in their substrate specificity and inhibitor binding profiles?

NUDT14 and NUDT5 share some similarities in substrate preference but exhibit notable differences in their structural features and inhibitor binding profiles:

• Substrate specificity:

  • Both NUDT14 and NUDT5 hydrolyze free ADP-ribose

  • NUDT14 has more pronounced activity toward UDP-glucose, converting it to glucose 1-phosphate and UMP

  • NUDT5 shows broader substrate specificity within the NUDIX family

• Structural comparison:

  • Crystal structure superimposition of NUDT5 and NUDT14 reveals conserved core structures but important differences

  • Both enzymes contain π stacking residues (Y17 and W34 in NUDT14; Y36 and W46 in NUDT5)

  • In NUDT5, inhibitor compound "9" is sandwiched between W46 and W28, whereas the binding mode differs in NUDT14

  • The hydrogen bonding network also differs: D35 in NUDT14 corresponds to E47 in NUDT5

• Inhibitor selectivity determinants:

  • The R51 residue in NUDT5 is not conserved in NUDT14 and appears critical for establishing ligand H-bond interactions

  • This structural difference explains why some inhibitors (e.g., TH5427) show exquisite selectivity for NUDT5 over NUDT14

  • The hydrophobic binding pocket differs, with NUDT14 utilizing L107 for interaction with aromatic groups of inhibitors

Understanding these differences is crucial for designing selective inhibitors and for interpreting the distinct physiological roles of these related enzymes.

What crystallization conditions have successfully yielded high-resolution structures of NUDT14?

Obtaining high-quality crystal structures of NUDT14 has been challenging, particularly for resolving the N-terminal domain. Recent advances have yielded significant insights:

• Co-crystallization with inhibitors - The breakthrough 1.82 Å resolution structure of NUDT14 was achieved through co-crystallization with a dual NUDT5/NUDT14 inhibitor, revealing the previously unresolved N-terminal domain structure . This suggests that small-molecule stabilization can facilitate crystallization.

• Protein construct optimization - The full-length construct including the N-terminal domain (residues 1-222) has been successfully crystallized when expressed with an N-terminal His-tag and purified to high homogeneity .

• Structure determination insights - The successful structure determination at 1.82 Å resolution provides sufficient detail to:

  • Resolve the intertwined β-sheets of the N-terminal domain with the NUDIX domain of the second subunit

  • Identify key residues in the active site (W34, Y17, D35, L107)

  • Characterize π–π stacking interactions and hydrogen bonds with bound ligands

Researchers seeking to crystallize NUDT14 should consider these conditions as a starting point, recognizing that co-crystallization with suitable ligands may be particularly effective for resolving the complete structure.

What are the optimal conditions for measuring NUDT14 enzymatic activity and inhibition in vitro?

Establishing reliable enzymatic assays for NUDT14 requires careful optimization of reaction conditions:

• Buffer composition:

  • 20mM Tris-HCl buffer (pH 8.0) containing 0.1M NaCl

  • 10% glycerol to enhance protein stability

  • 1mM DTT to maintain reduced cysteine residues

  • Magnesium as essential cofactor, as NUDT14 binds magnesium for its catalytic activity

• Substrate considerations:

  • UDP-glucose or ADP-ribose as primary substrates, as NUDT14 hydrolyzes ADP-ribose into ribose 5-phosphate and AMP, and UDP-glucose to glucose 1-phosphate and UMP

  • Substrate concentration at or near Km for inhibition studies

• Detection methods:

  • Malachite green assay for phosphate release

  • HPLC separation and quantification of reaction products

  • Coupled enzyme assays

  • Mass spectrometry for direct product analysis

• Inhibition assay design:

  • Pre-incubation of enzyme with inhibitor before substrate addition

  • Controls for compound interference with detection methods

  • Generation of complete inhibition curves (IC₅₀ determination)

For inhibitor characterization, SPR has been successfully applied to measure direct binding to NUDT14 with affinities in the 400 nM range for dual NUDT5/NUDT14 inhibitors .

What experimental approaches can determine if NUDT14 plays a role in metabolic regulation beyond nucleotide metabolism?

Investigating broader metabolic roles of NUDT14 beyond its established nucleotide metabolism function requires systematic approaches:

• Metabolomic profiling:

  • Untargeted metabolomics following NUDT14 knockdown/overexpression to identify affected metabolic pathways

  • Stable isotope-labeled metabolite tracing to track carbon flux through different pathways

  • Targeted analysis of UDP-sugar metabolism, which is relevant given NUDT14's activity on UDP-glucose

• Genetic manipulation models:

  • CRISPR/Cas9-mediated knockout in cell lines and animal models

  • Inducible expression systems to study acute vs. chronic effects

  • Tissue-specific conditional knockouts to address potential developmental compensation

• Protein-protein interaction network analysis:

  • Proximity labeling approaches to identify neighbors in metabolic complexes

  • Immunoprecipitation coupled with mass spectrometry

  • Two-hybrid screening with metabolic enzymes, a technique already validated for NUDT14 interactions

The interaction between NUDT14 and UDP-glucose suggests potential roles in glycosylation pathways, which are critical for protein modification and function. This represents a promising area for investigation beyond canonical nucleotide metabolism.

How does NUDT14 interact with viral proteins, and what are the implications for antiviral research?

NUDT14 has been shown to interact with viral proteins, particularly the human cytomegalovirus (HCMV) RL13 protein, with important implications for viral replication:

• Interaction characterization:

  • Two-hybrid screening has provided direct evidence for specific interaction between HCMV RL13 and host NUDT14

  • GST pull-down assays and co-immunoprecipitation in HEK293 cells have confirmed this interaction

  • Fluorescence confocal microscopy has demonstrated co-localization of RL13 protein with NUDT14 in the cell membrane and cytoplasm

• Functional consequences:

  • Decreasing NUDT14 expression via siRNAs increases viral DNA copies in HCMV-infected cells

  • This suggests NUDT14 may normally play a restrictive role in viral replication

  • Interestingly, overexpression of NUDT14 does not significantly affect viral DNA levels, indicating complex regulatory mechanisms

• Research implications:

  • NUDT14 may represent a host restriction factor that viruses must overcome

  • The interaction with viral proteins suggests NUDT14 could be involved in innate immune responses

  • Based on NUDT14's known functions, this interaction may affect viral DNA replication

These findings suggest that NUDT14 may offer potential in the modulation of viral infection, opening new avenues for antiviral research strategies targeting host-virus protein interactions.

What cellular models best represent physiological NUDT14 function for in vitro studies?

Selecting appropriate cellular models is crucial for studying physiological NUDT14 function:

• Cell lines with documented endogenous NUDT14 expression:

  • HEK293 cells have been successfully used for NUDT14 interaction studies

  • U2OS ARH3 KO cells have been employed for ADP-ribosylation studies related to NUDT14 function

  • Cancer cell lines with elevated NUDT14 expression, as NUDT14 is frequently expressed at elevated levels in certain cancer cell types

• Genetic modification approaches:

  • CRISPR/Cas9 knockout cell lines to study loss-of-function effects

  • Inducible expression systems for controlled NUDT14 overexpression

  • Knock-in of tagged NUDT14 (HiBiT, fluorescent proteins) for tracking studies

• Disease-relevant models:

  • For viral infection studies: Cell lines permissive to HCMV infection, which have provided valuable insights into the functional interaction between NUDT14 and viral proteins

  • For metabolic disorders: Cell types with active UDP-glucose metabolism, given NUDT14's role as a UDP-glucose pyrophosphatase

• Specialized model systems:

  • 3D culture systems to better mimic tissue architecture

  • Co-culture models to study cell-cell interactions

  • Validation of key findings in primary human cells when feasible

When selecting cellular models, researchers should consider NUDT14 expression levels, relevant metabolic pathways, and the specific research question being addressed.

How can researchers effectively design primers and probes for NUDT14 genetic analysis?

Designing specific primers and probes for NUDT14 genetic analysis requires careful consideration of sequence specificity:

• NUDT14 reference sequence information:

  • mRNA RefSeq: NM_177533

  • Protein RefSeq: NP_803877

  • Gene ID: 256281

  • Chromosome location: 14q32.33

• Primer design considerations:

  • Target unique regions with minimal homology to other NUDIX family members

  • Inclusion of exon-exon junctions in RT-PCR primers to avoid genomic DNA amplification

  • Utilization of NUDT14-specific 5' or 3' UTR regions when possible

• Validation strategies:

  • Sequencing of PCR products to confirm correct amplification

  • Use of positive and negative control templates

  • Melt curve analysis in qPCR to confirm single product amplification

  • Testing primers in cell lines with known NUDT14 expression levels

• Specialized applications:

  • For CRISPR guide RNA design, target unique sequences with minimal off-target potential

  • For gene expression analysis, consider primers that can detect alternative splicing variants if relevant

These considerations will help ensure specific detection of NUDT14 while avoiding cross-reactivity with related sequences.

What are the best methodologies for screening potential NUDT14 substrates beyond known examples?

Discovering new NUDT14 substrates requires systematic screening approaches:

• In vitro substrate screening:

  • Activity-based screening of nucleotide sugar libraries

  • NMR-based metabolite screening to detect hydrolysis products

  • Mass spectrometry to identify reaction products from complex metabolite mixtures

  • High-throughput colorimetric assays detecting generic hydrolysis products (e.g., phosphate)

• Structural prediction approaches:

  • Molecular docking of potential substrates into the NUDT14 active site, now feasible with the resolved crystal structure

  • Pharmacophore modeling based on known substrates (UDP-glucose, ADP-ribose)

  • Structure-based virtual screening of metabolite databases

• Metabolomic approaches:

  • Untargeted metabolomics in NUDT14 knockout vs. wildtype cells

  • Stable isotope labeling to track conversion of potential substrates

  • Correlation analysis between NUDT14 expression and metabolite levels across cell types

• Thermal shift assays:

  • Differential scanning fluorimetry with potential substrates

  • Cellular thermal shift assay (CETSA) with metabolite treatment

  • These methods detect stabilization of NUDT14 upon substrate binding

Given the known activity of NUDT14 on UDP-glucose and ADP-ribose , other nucleotide sugars and ADP-derivatives represent logical candidates for expanded substrate screening.