NUDT5 Antibody

What is NUDT5 and why is it important in research?

NUDT5 is a member of the NUDIX hydrolases superfamily that functions as an important enzyme in nucleotide metabolism . It exhibits dual enzymatic functions: it can act as an ADP-sugar pyrophosphatase in the absence of diphosphate, or catalyze the synthesis of ATP in the presence of diphosphate . Recent studies have highlighted NUDT5's significance in regulating nuclear ATP dynamics and ADPR-related metabolism functions in ER-positive breast cancer cells . Additionally, NUDT5 has been associated with the prognosis of breast cancer, lung cancer, and prostate cancer, making it an important target for cancer research . The enzyme has garnered significant attention due to its overexpression in triple-negative breast cancer (TNBC) and its potential as a therapeutic target .

What are the primary substrates of NUDT5?

NUDT5 can hydrolyze various modified nucleoside diphosphates with similar activities. Its primary substrates include:

• ADP-ribose (ADPR)

• ADP-mannose

• ADP-glucose

• 8-oxo-GDP

• 8-oxo-dGDP

These hydrolysis activities have been confirmed through multiple studies . The enzyme can also hydrolyze other nucleotide sugars, albeit with lower activity . In the presence of diphosphate, NUDT5 mediates the synthesis of ATP in the nucleus by catalyzing the conversion of ADP-ribose to ATP and ribose 5-phosphate, particularly when dephosphorylated at Thr-45 .

How can NUDT5 protein expression be detected in research samples?

NUDT5 protein expression can be detected using several techniques:

• Immunohistochemistry (IHC-P): Using specific NUDT5 antibodies like rabbit polyclonal antibodies that react with human samples . This method is particularly useful for tissue sections.

• Immunocytochemistry/Immunofluorescence (ICC/IF): NUDT5 antibodies suitable for ICC/IF can be used to visualize the protein in cultured cells .

• Western Blotting: As demonstrated in research studies, pull-down experiments followed by Western blotting using specific NUDT5 antibodies can confirm the presence of endogenous NUDT5 .

• Chemoproteomics: Advanced approaches using affinity probes like CBH-003 (compound 7) can be used to confirm binding to endogenous NUDT5 proteins .

When selecting antibodies for these applications, researchers should ensure they are validated for the specific application and species of interest, such as antibodies targeting recombinant fragments within human NUDT5 amino acids 1-200 .

What methods are available for measuring NUDT5 enzymatic activity?

Several validated assays are available for measuring NUDT5 enzymatic activity:

• Transcreener NUDT5 Assay: This assay directly detects AMP produced by NUDT5 to measure enzymatic activity. It uses an antibody selective to AMP over a far-red fluorescent tracer. AMP produced in the reaction competes with the tracer, changing the fluorescent properties and providing a fluorescent readout .

• AMP-Glo Assay: This assay monitors the NUDT5-mediated conversion of ADPr into AMP and ribose-5-phosphate. It has been used in screening studies to identify inhibitors like ibrutinib .

• NanoBRET Target Engagement Assay: This live-cell assay can measure the engagement of inhibitors with NUDT5. It typically uses a combination of NanoLuc-NUDT5 fusion proteins and BODIPY-labeled probes .

The Transcreener NUDT5 Assay is available in both FP and TR-FRET formats and offers a simple mix-and-read protocol. The assay demonstrates excellent performance with Z' values of 0.91 under optimized conditions, making it suitable for high-throughput screening .

How does NUDT5 contribute to nuclear ATP synthesis?

NUDT5 plays a crucial role in nuclear ATP synthesis through the following mechanism:

• When dephosphorylated at Thr-45, NUDT5 mediates the synthesis of ATP in the nucleus by catalyzing the conversion of ADP-ribose to ATP and ribose 5-phosphate in the presence of diphosphate .

• This nuclear ATP generation is required for extensive chromatin remodeling events that are energy-consuming .

• Recent studies have highlighted the significance of NUDT5 in regulating nuclear ATP dynamics and ADPR-related metabolism functions, particularly in ER-positive breast cancer cells .

The ability of NUDT5 to generate ATP within the nucleus provides a localized energy source for nuclear processes, particularly those involved in chromatin remodeling. This function has significant implications for gene expression regulation and DNA-dependent processes that require ATP, making NUDT5 an important player in nuclear metabolism .

What is the relationship between NUDT5 and oxidative DNA damage?

NUDT5 plays a significant role in regulating oxidative DNA damage, particularly in triple-negative breast cancer (TNBC) cells:

• Loss of NUDT5 results in an increase in the oxidative DNA lesion 8-oxoG and triggers DNA damage response in the nucleus .

• NUDT5 can hydrolyze 8-oxo-GDP and 8-oxo-dGDP, which are oxidized forms of guanine nucleotides that can cause mutations if incorporated into DNA .

• The loss or inhibition of NUDT5 leads to interference with DNA replication, ultimately inhibiting cell proliferation .

• Studies have demonstrated that NUDT5 is crucial for preventing oxidative DNA damage in TNBC cells, and its loss or inhibition significantly suppresses the growth of TNBCs .

These findings suggest that NUDT5 functions as a protective enzyme against oxidative DNA damage by removing oxidized nucleotides from the nucleotide pool, preventing their incorporation into DNA during replication. This protective function is particularly important in cancer cells, which often have elevated levels of reactive oxygen species .

How do NUDT5 inhibitors affect cancer cell growth and DNA replication?

NUDT5 inhibitors have significant effects on cancer cell growth and DNA replication, particularly in triple-negative breast cancer (TNBC):

• Growth Suppression: Loss of NUDT5 through inhibition results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is primarily attributed to the suppression of proliferation rather than induction of cell death .

• DNA Replication Interference: NUDT5 inhibition leads to interference with DNA replication fork progression. This is evidenced by measurements of DNA fiber length, which show altered replication dynamics when NUDT5 is inhibited .

• Oxidative DNA Damage Accumulation: Treatment with NUDT5 inhibitors like TH5427 leads to an increase in the oxidative DNA lesion 8-oxoG and triggers DNA damage response markers such as γH2AX .

• Mechanistic Pathway: The mechanism appears to involve:

  • Accumulation of oxidized nucleotides in the nucleotide pool

  • Incorporation of these damaged nucleotides into DNA during replication

  • Activation of DNA damage response pathways

  • Resulting inhibition of cell proliferation

These findings provide a mechanistic basis for the potential therapeutic application of NUDT5 inhibitors in TNBC and potentially other cancers where NUDT5 is overexpressed .

What are the structural insights into NUDT5 inhibition by small molecules?

Recent structural studies have provided valuable insights into NUDT5 inhibition by small molecules:

• Unexpected Inhibitors: BTK inhibitors like ibrutinib (compound 1) have been identified as unexpected, noncovalent, off-target inhibitors of NUDT5. This discovery was made through screening a small kinase inhibitor library .

• Binding Mode: X-ray crystallography revealed an unusual binding mode that is independent of the reactive acrylamide warhead present in BTK inhibitors. This binding involves interactions with the adenine moiety, with strong π-π stacking interactions observed in the enzymatic pocket .

• Structure-Activity Relationships (SAR): By exploring structure-activity relationships around the core scaffold of ibrutinib, researchers identified a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor (compound 9) .

• Crystal Structures: Cocrystal structures of NUDT5 and NUDT14 with inhibitors have been resolved, providing valuable insights for the development of selective inhibitors. These structures reveal differences in the binding pockets that can be exploited for selective targeting .

• Cofactor Mimicry: The inhibition mechanism involves cofactor mimicry, which represents an attractive strategy for enzyme inhibitor development but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome .

These structural insights are crucial for the rational design of selective NUDT5 inhibitors that could be developed into chemical probes or potential therapeutic agents .

What methodological approaches are optimal for studying NUDT5 target engagement in living cells?

Several sophisticated methodological approaches have been developed for studying NUDT5 target engagement in living cells:

• NanoBRET Target Engagement Assay:

  • This approach uses N- or C-terminal NUDT5-NanoLuc fusions expressed in cells.

  • An energy-transfer fluorescent probe (ETF) like CBH-004 (compound 8) is added, which binds to NUDT5.

  • When an inhibitor engages with NUDT5, it displaces the probe, resulting in a change in bioluminescence resonance energy transfer (BRET) signal.

  • This assay has been optimized with a 2.5 nM probe concentration using N-terminal NanoLuc-NUDT5 fusion .

• Pull-down Experiments with Affinity Probes:

  • Affinity probes like CBH-003 (compound 7) with a free primary amine can be used for pull-down experiments.

  • These probes can be designed based on cocrystal structures of known NUDT5 inhibitors.

  • After pull-down, Western blotting with specific NUDT5 antibodies confirms target engagement .

• Chemoproteomic Approaches:

  • These approaches can provide a proteome-wide view of inhibitor binding.

  • They can help assess the selectivity of inhibitors across the proteome and identify potential off-targets .

• HiBiT CETSA (Cellular Thermal Shift Assay):

  • This technique can confirm inhibitor target engagement in live cells by measuring thermal stabilization of the target protein upon inhibitor binding .

These methods provide complementary information about inhibitor engagement with NUDT5 in cellular contexts and are essential for validating potential chemical probes or therapeutic candidates targeting NUDT5 .

How can researchers optimize experimental conditions for NUDT5 activity assays?

Optimizing experimental conditions for NUDT5 activity assays requires careful consideration of several parameters:

• Substrate Selection and Concentration:

  • NUDT5 can use different substrates including ADP-ribose (ADPR), ADP-mannose, and ADP-glucose.

  • For initial velocity conditions, ADPR at 100 μM starting concentration has been shown to be effective .

  • Substrate concentration should be optimized based on the Km value for the specific substrate being used.

• Reaction Time and Temperature:

  • Optimal enzyme reactions typically occur for 60 minutes at room temperature .

  • Time course experiments should be performed to ensure measurements are made within the linear range of the reaction.

• Buffer Conditions:

  • The use of appropriate buffers like Tris solution is crucial for maintaining optimal enzyme activity .

  • pH, salt concentration, and the presence of divalent cations (like Mg2+) should be optimized.

• Detection Method Selection:

  • The Transcreener NUDT5 Assay is available with Fluorescence Polarization (FP) and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) readouts .

  • The choice between these should be based on the available instrumentation and specific research requirements.

• Assay Validation and Quality Control:

  • Z' measurements using optimized NUDT5 reaction conditions should indicate a robust assay (Z' values > 0.7) .

  • Standard curves using AMP should be used to convert raw data to actual AMP produced in the reaction .

• Plate Format Optimization:

  • The assay should be compatible with 96, 384, and 1536-well formats, allowing flexibility based on throughput requirements .

By carefully optimizing these parameters, researchers can achieve reliable and reproducible measurements of NUDT5 activity, which is critical for inhibitor screening and mechanistic studies .

What are the best practices for validating NUDT5 antibody specificity in research applications?

Validating NUDT5 antibody specificity is crucial for ensuring reliable experimental results. Here are best practices:

• Multiple Antibody Validation Approaches:

  • Western blotting with recombinant NUDT5 protein as a positive control

  • Immunoprecipitation followed by mass spectrometry identification

  • Use of NUDT5 knockout or knockdown samples as negative controls

  • Cross-validation with multiple antibodies targeting different epitopes

• Epitope Selection:

  • Choose antibodies targeting unique regions of NUDT5, such as those within amino acids 1-200 of the human protein .

  • Avoid epitopes with high sequence similarity to other NUDIX family members, particularly NUDT14, which shares functional similarities with NUDT5 .

• Specificity Testing:

  • Perform Western blot analysis across multiple cell lines with known NUDT5 expression levels.

  • Include related NUDIX family proteins (especially NUDT14) as controls to test for cross-reactivity.

  • For immunohistochemistry applications, include tissue panels with variable NUDT5 expression.

• Functional Validation:

  • Use affinity probes like CBH-003 (compound 7) for pull-down experiments followed by Western blotting with the NUDT5 antibody .

  • Confirm results with chemoproteomics approaches to verify binding specificity .

• Application-Specific Validation:

  • For IHC-P: Validate using tissues with known NUDT5 expression patterns

  • For ICC/IF: Confirm subcellular localization patterns match known NUDT5 distribution

  • For co-IP: Validate with mass spectrometry to confirm pulled-down proteins

• Documentation and Reporting:

  • Document all validation steps performed

  • Include validation data in publications

  • Report antibody catalog numbers, lot numbers, and dilutions used

By following these best practices, researchers can ensure that their NUDT5 antibody-based experiments produce reliable and reproducible results that accurately reflect NUDT5 biology .