NME1 Human, Active

What is NME1 and what is its primary function?

NME1, also known as Nucleoside Diphosphate Kinase A (NDPK-A) or Nm23-H1, is an enzyme encoded by the NME1 gene in humans. It was identified as the first metastasis suppressor gene, exhibiting reduced expression in highly metastatic cells . The primary function of NME1 is its nucleoside diphosphate kinase activity, which balances intracellular pools of nucleotide diphosphates and triphosphates . This enzymatic function plays critical roles in regulating cytoskeletal rearrangement, cell motility, endocytosis, intracellular trafficking, and metastasis suppression .

To study the primary function of NME1, researchers should consider employing activity assays that measure phosphotransferase activity. The standard approach involves a coupled system with pyruvate kinase and lactate dehydrogenase, where specific activity can be defined as the amount of enzyme that converts 1.0 μmole each of ATP and TDP to ADP and TTP per minute at pH 7.5 at 25°C .

What is the structural organization of NME1?

NME1 forms a homo-hexameric structure stabilized by cross-interaction between the Kpn-loop region and the neighboring C-terminal domain . This oligomerization into a hexameric structure is crucial for its phosphotransferase activity, including NDP phosphorylation, protein-histidine phosphorylation, and metastasis suppression .

Recombinant human NME1 is a single, non-glycosylated polypeptide chain containing 152 amino acids with a molecular mass of approximately 17.1 kDa . The protein can also exist as heterohexamers with other NME proteins, particularly NME2, with which it shares 88% sequence homology .

Researchers investigating NME1 structure should employ techniques such as X-ray crystallography, cryo-electron microscopy, or small-angle X-ray scattering (SAXS) to visualize the hexameric assembly and understand how structural changes affect function.

How does NME1 expression correlate with cancer progression?

NME1 expression levels typically demonstrate an inverse correlation with metastatic potential in several cancer types. Low NME1 levels are associated with aggressive carcinomas including colon, breast, gastric, and melanoma cancers . Interestingly, high levels of NME1 have been linked to advanced thyroid cancer, suggesting tissue-specific roles .

A 2023 bioinformatics study suggested that the NME1 gene might have a prognostic role in neuroblastoma . Research has shown that mutations in this gene have been identified in aggressive neuroblastomas .

For studies examining NME1 expression in cancer progression, researchers should employ multiple approaches:

• qRT-PCR for mRNA expression analysis

• Western blotting for protein quantification

• Immunohistochemistry of patient samples

• Correlation of expression with clinical outcomes and metastatic status

What are the multiple enzymatic activities associated with NME1?

NME1 is a multifunctional enzyme with several distinct catalytic activities:

• Nucleoside Diphosphate Kinase (NDPK) Activity: The primary function involving the transfer of phosphate from nucleoside triphosphates to nucleoside diphosphates .

• Histidine/Aspartic Acid-Specific Protein Kinase Activity: NME1 can phosphorylate histidine and aspartic acid residues on target proteins .

• 3′–5′ Exonuclease Activity: This activity suggests roles in DNA proofreading and repair .

• Geranyl/Farnesyl Pyrophosphate Kinase Activity: This function implicates NME1 in additional metabolic pathways .

To investigate these different enzymatic activities, researchers should develop specific assays for each function. For example, the exonuclease activity can be assessed using labeled oligonucleotide substrates, while protein kinase activity requires phosphorylation assays with appropriate protein substrates.

What mechanisms underlie NME1's metastasis suppression function?

NME1 suppresses metastasis through multiple mechanisms:

• Regulation of Small GTPases: NME1 negatively regulates Rac1 and Cdc42 by interacting with their specific guanine nucleotide exchange factors Tiam1 and Dbl-1, respectively . It also interacts with Rad protein to regulate Rad GDP/GTP cycling, negatively regulating downstream molecules responsible for cytoskeletal organization and cell motility .

• Modulation of Gene Expression: NME1 has been demonstrated to directly bind to promoter regions of target genes and activate transcription. For example, it can bind to the ALDOC gene promoter, inducing transcriptional changes that affect metastatic potential . NME1 binding to the promoter results in enhanced occupancy, increased presence of epigenetic activation markers (H3K4me3 and H3K27ac), and recruitment of RNA polymerase II .

• Interference with TGF-β Signaling: NME1 may regulate TGF-β signaling through interaction with serine/threonine kinase receptor-associated protein (STRAP) via an intermolecular disulfide bond . This is significant as TGF-β signaling is a dominant pathway triggering invasion and metastasis in advanced cancer cells.

• Regulation of Metastasis-Related Genes: NME1 inhibits cell motility by regulating expression of proteins involved in invasion and metastasis, such as lysophosphatidic acid receptor EDG2 and matrix metalloproteinase MMP2 .

To study these mechanisms, researchers should combine molecular approaches (ChIP-seq, RNA-seq), protein interaction studies (co-IP, proximity ligation), and functional assays (cell migration, invasion) in appropriate model systems.

How is NME1 affected by oxidative stress and metabolic conditions?

NME1 structure and function are modulated by reactive oxygen species (ROS) via three redox-sensitive cysteine residues . Under oxidative stress, an intramolecular disulfide bridge forms between Cys4 and Cys145, triggering conformational rearrangement that destabilizes the hexameric state and leads to dimer formation .

Recent research has identified NME1 as a major coenzyme A (CoA) binding protein in cultured cells and rat tissues . NME1 can be covalently modified by CoA (CoAlation) at Cys109 under disulfide stress conditions . This modification inhibits the NDPK activity of NME1, and the inhibition is reversible by reducing agents like DTT . Additionally, CoA can function as a competitive ATP-binding inhibitor of NME1's NDPK activity .

These findings reveal a novel regulatory mechanism where NME1 is modulated by the key metabolic integrator CoA, particularly pronounced during cellular responses to oxidative or metabolic stress .

Methodological approaches to study these modifications include:

• Site-directed mutagenesis of cysteine residues

• Mass spectrometry to detect CoAlation

• Enzymatic assays under varying redox conditions

• Structural studies comparing native and modified forms

What techniques are most effective for studying NME1's role in transcriptional regulation?

To investigate NME1's transcriptional regulatory functions, researchers should employ the following approaches:

• Chromatin Immunoprecipitation (ChIP): This technique has successfully demonstrated NME1 recruitment to the promoter region of target genes like ALDOC . ChIP followed by sequencing (ChIP-seq) can identify genome-wide binding sites.

• Promoter-Luciferase Assays: These constructs have been employed to measure the impact of NME1 on transcriptional activity of target genes . This approach allows quantitative assessment of NME1's effect on specific promoters.

• Analysis of Epigenetic Markers: Examination of histone modifications such as H3K4me3 and H3K27ac at NME1-regulated promoters can reveal mechanisms of transcriptional activation .

• RNA Polymerase II Recruitment Studies: Assessment of RNA polymerase II occupancy at target promoters provides insights into transcription initiation and elongation processes influenced by NME1 .

• Pre-mRNA Measurement: Quantifying pre-mRNA levels of target genes can confirm direct transcriptional effects rather than post-transcriptional regulation .

A comprehensive approach combining these techniques with gene expression profiling (RNA-seq) and functional validation can provide robust evidence for NME1's transcriptional regulatory mechanisms.

How can researchers distinguish between the functions of NME1 and NME2?

• Isoform-Specific Knockdown/Knockout: Using siRNA, shRNA, or CRISPR-Cas9 to selectively target either NME1 or NME2.

• Focus on Unique Regions: The ten amino acids that differ between NME1 and NME2 may be responsible for their distinct functions . Structure-function studies focusing on these regions can help identify isoform-specific activities.

• Study of Alternative Splice Variants: NME1 gene encodes two proteins (NME1;152aa and NME1L;177aa) by alternative splicing, with NME1L containing 25 additional N-terminal amino acids . This distinction may contribute to functional differences.

• Protein-Protein Interaction Analysis: Identification of protein binding partners unique to each isoform can elucidate distinct functional pathways.

• Metastasis Models: While NME1 has well-documented anti-metastatic activity, NME2 appears to lack this function . Comparative studies in metastasis models can highlight these differences.

These methodological approaches, combined with careful experimental design and validation, can help researchers delineate the distinct biological roles of these highly similar proteins.

What experimental models are most appropriate for studying NME1 in cancer research?

For comprehensive investigation of NME1 in cancer research, multiple experimental models should be employed:

For metastasis studies specifically, researchers should note that ablation of the Nme1-Nme2 locus in mice confers strong metastatic activity in a model of ultraviolet light-induced melanoma, providing in vivo validation of the metastasis suppressor function .

What methods are recommended for producing and purifying active NME1 for in vitro studies?

For researchers requiring active NME1 protein for in vitro experiments, the following methodological guidelines are recommended:

• Expression System: Recombinant human NME1 is commonly produced in E. coli as a single, non-glycosylated polypeptide chain containing 152 amino acids with a molecular mass of 17.1kDa .

• Purification: Standard purification approaches include affinity chromatography (if tagged) followed by size exclusion chromatography to isolate the hexameric form.

• Buffer Conditions: Optimal storage conditions include a buffer containing 10% glycerol, 20mM Tris-HCl (pH 7.5), and 1mM DTT to maintain stability .

• Storage: For short-term use (2-4 weeks), the protein can be stored at 4°C. For longer periods, storage at -20°C is recommended with the addition of a carrier protein (0.1% HSA or BSA) to prevent activity loss . Multiple freeze-thaw cycles should be avoided.

• Quality Control: Purity should be >90% as determined by SDS-PAGE . Activity assays should confirm specific activity >1,200 units/mg, defined as the amount of enzyme that converts 1.0 μmole each of ATP and TDP to ADP and TTP per minute at pH 7.5 at 25°C in a coupled system with pyruvate kinase/lactate dehydrogenase .

These standardized protocols ensure production of active NME1 suitable for enzymatic, structural, and functional studies.

How can researchers effectively measure the diverse enzymatic activities of NME1?

Given NME1's multiple enzymatic functions, researchers should employ specific assays for each activity:

• Nucleoside Diphosphate Kinase Activity:

  • Coupled enzymatic assay with pyruvate kinase and lactate dehydrogenase

  • Direct measurement of nucleotide conversion by HPLC

  • Radioactive assays using labeled nucleotides

• 3′–5′ Exonuclease Activity:

  • Labeled oligonucleotide substrates followed by gel electrophoresis

  • Fluorescence-based real-time assays with quenched substrates

• Protein-Histidine Kinase Activity:

  • Phosphorylation assays with target proteins

  • Western blotting with phospho-histidine specific antibodies

  • Mass spectrometry to identify phosphorylation sites

• Activity Modulation Studies:

  • Effects of oxidizing agents on activity

  • CoA binding and inhibition assays

  • Structural studies correlating hexamer/dimer status with activity

When performing these assays, researchers should carefully control experimental conditions, particularly redox status, as the enzymatic activities of NME1 are sensitive to oxidative modifications of cysteine residues .

What strategies are most effective for studying NME1-mediated transcriptional regulation?

For researchers investigating NME1's role in transcriptional regulation, a multi-faceted approach is recommended:

• Target Gene Identification:

  • RNA-seq comparing NME1-expressing versus NME1-depleted cells

  • ChIP-seq to identify genome-wide NME1 binding sites

  • Motif analysis to identify potential DNA binding preferences

• Mechanistic Studies:

  • Promoter-luciferase constructs to quantify transcriptional effects

  • ChIP assays to detect NME1 recruitment to specific promoters

  • Analysis of histone modifications (H3K4me3, H3K27ac) and RNA polymerase II recruitment

  • Assessment of pre-mRNA levels to confirm direct transcriptional effects

• Functional Validation:

  • Mutational analysis of NME1 to identify domains required for transcriptional regulation

  • Rescue experiments restoring expression of NME1-regulated genes

  • Correlation of transcriptional changes with metastatic phenotypes

The case study of NME1 regulation of ALDOC provides a methodological template, demonstrating that NME1 enhances transcription through direct binding to the promoter region, increased presence of epigenetic activation markers, and recruitment of RNA polymerase II .

What are the unresolved questions regarding NME1's role in cancer progression?

Despite extensive research, several key questions about NME1 remain unanswered:

• Cancer Type Specificity: Why does NME1 function as a metastasis suppressor in some cancers but show different effects in others, such as advanced thyroid cancer ?

• Integration of Multiple Functions: How do NME1's diverse enzymatic activities (NDPK, exonuclease, protein kinase) collectively contribute to its metastasis suppression function?

• Clinical Translation: Can NME1 expression or activity be effectively targeted for therapeutic benefit in cancer patients?

• Interplay with Metabolism: How does the newly discovered interaction with CoA and sensitivity to metabolic stress influence NME1's role in tumor progression ?

• Regulatory Mechanisms: What factors control NME1 expression, localization, and activity in different cellular contexts?

Addressing these questions requires integrative approaches combining molecular, cellular, and in vivo studies with clinical correlations.

How can researchers leverage new technologies to advance NME1 research?

Emerging technologies offer opportunities to address longstanding questions about NME1:

• Single-Cell Analysis: Single-cell RNA-seq and proteomics can reveal heterogeneity in NME1 expression within tumors and correlate with metastatic potential at the cellular level.

• CRISPR-Based Technologies: CRISPR screens can identify synthetic lethal interactions with NME1 loss or potential compensatory mechanisms. CRISPR activation/inhibition systems can modulate NME1 expression with temporal control.

• Proximity Labeling: Techniques like BioID or APEX can identify NME1 protein interaction networks in specific cellular compartments, including the nucleus where it functions in transcriptional regulation .

• Live-Cell Imaging: Advanced microscopy combined with optogenetic tools can visualize NME1 dynamics during cellular processes relevant to metastasis.

• Structural Biology Advances: Cryo-EM can provide insights into NME1 complexes with interacting proteins or DNA, informing structure-based drug design.

These technological approaches, combined with computational integration of multi-omics data, can provide new insights into NME1 function and potentially identify novel therapeutic strategies targeting NME1 pathways.