NME1 Human

What is NME1 and what is its primary function?

NME1, also called NM23-H1, is the first identified metastasis suppressor gene (MSG). It inhibits different biological processes during metastatic progression without globally influencing primary tumor development. NME1 belongs to a family of proteins with evolutionarily conserved nucleoside diphosphate kinase (NDPK) activity . The primary function of NME1 is to suppress metastatic activity in cancer cells while showing reduced expression in metastatic cancers .

As a member of Group I NME genes (NME1-4), it possesses NDPK activity, which is critical for its function. The protein exhibits multiple biological roles (hence its classification as a "moonlighting protein"), including inhibition of cell migration, modulation of transcription, and participation in various signaling pathways .

How many NME homologs exist and how are they classified?

The human genome encodes ten NME (NM23) homologs that are divided into two distinct groups based on sequence homology and enzymatic activity:

• Group I: Includes NME1-4 (NM23-H1-H4), which all possess nucleoside diphosphate kinase (NDPK) activity and share high sequence homology

• Group II: Comprises NME5-9 (NM23-H5-H9) and RP2 (retinitis pigmentosa 2), which are more divergent in sequence and exhibit little to no NDPK activity

This classification is important for understanding the functional diversity within the NME family and for designing targeted research approaches.

What is the correlation between NME1 expression and cancer prognosis?

The relationship between NME1 expression and cancer prognosis varies by cancer type:

• Inverse correlation with metastatic potential (better prognosis with higher expression): Melanomas and epithelial tumors including breast, liver, colon, and cervical carcinomas

• Positive correlation with poor prognosis (worse outcomes with higher expression): Hematological malignancies, ovarian cancer, prostate cancer, and neuroblastoma

This context-dependent relationship highlights the complex role of NME1 in different cellular environments and underscores the importance of tissue-specific research when studying NME1 as a potential biomarker or therapeutic target.

How does NME1 function as a transcriptional regulator?

NME1 has been demonstrated to function as a direct transcriptional regulator. Studies show that NME1 can:

• Directly bind to DNA motifs in promoter regions of various genes, including PDGFA, TP53, and ALDOC

• Enhance gene transcription through direct binding to promoter regions, as evidenced with the ALDOC gene where NME1:

  • Increases expression of ALDOC pre-mRNA

  • Activates the ALDOC promoter-luciferase module

  • Is physically detected at the ALDOC promoter

  • When forcibly expressed, enhances occupancy at the promoter

• Induce epigenetic activation markers at target promoters:

  • Increases presence of H3K4me3 (trimethylation of lysine 4 on histone 3)

  • Enhances H3K27ac (acetylation of lysine 27 on histone 3)

  • Recruits RNA polymerase II to promote transcription

This transcriptional regulatory function represents a significant mechanism through which NME1 may exert its metastasis suppressor activity.

What are the mechanisms by which NME1 inhibits cell migration and metastasis?

NME1 inhibits cell migration and metastasis through several interconnected mechanisms:

• Negative regulation of Rho-Rac signaling:

  • Inhibits Rac1 activity, a pleiotropic regulator of cell motility

  • Negatively regulates Tiam1 (a Rac1-specific nucleotide exchange factor), resulting in attenuated Rac1 activation

• Inhibition of invadopodia formation:

  • Suppresses formation of actin-driven membrane protrusions

  • Regulates MT1-MMP, the key invadopodial metalloproteinase involved in matrix degradation

• Transcriptional regulation:

  • Modulates expression of genes involved in metastatic processes

  • Directly binds to promoters of genes involved in cell motility and invasion

Understanding these mechanisms provides potential intervention points for developing anti-metastatic therapies targeting the NME1 pathway.

What is the structural basis for NME1's interaction with Coenzyme A (CoA)?

NME1 exhibits a unique mode of CoA binding that has been characterized through crystallography and molecular dynamics studies:

• Binding location: CoA binds non-covalently to the nucleotide-binding site of NME1

• Molecular dynamics analysis revealed multiple binding conformations:

  • Cluster 1: Na+ coordinates with β- and 3'-phosphates of CoA, with the pantetheine tail exposed to solvent

  • Cluster 2: Pantetheine tail stabilized through intramolecular interactions between amino groups, thiol group, and β-phosphate, with additional interactions with E93 and T94

  • Cluster 3: Pantetheine tail bends to establish hydrophobic-like interactions with V112 and T94

  • Cluster 4: Coordination of 3'-phosphate with Na+ induces a shift in R88, creating a pocket-like arrangement of L85, D121, and S122 where the pantetheine tail resides

• Critical residues: Mutation studies show that R58 and T94 are essential for CoA binding, as R58E and T94D mutations disrupt the interaction

This detailed structural understanding provides insights into the molecular basis of NME1's interaction with CoA and its potential regulatory implications.

What are the recommended methods for expressing and purifying recombinant NME1 protein?

For successful expression and purification of recombinant NME1, the following protocol is recommended based on published methodology:

• Expression system:

  • Use E. coli as the expression host

  • Transform with an appropriate expression vector containing the NME1 gene (with or without tags as needed)

  • Grow cultures at 37°C until OD600 reaches 0.8

  • Induce with 0.5 mM IPTG

  • Incubate overnight at 25°C for optimal protein expression

• Cell harvesting and lysis:

  • Harvest cells by centrifugation (15 min at 6200× g at 4°C)

  • Resuspend pellet in lysis buffer containing:

    • 50 mM Tris pH 7.5

    • 0.5 M NaCl

    • 5 mM imidazole

    • 1 mM beta-mercaptoethanol

    • 50 μg/mL DNase I

    • Protease inhibitor cocktail

    • 10 mM MgCl2

  • Lyse cells by sonication (15 cycles of 15s on/20s off)

  • Clear lysate by centrifugation (40 min at 39,000× g at 4°C)

• Purification:

  • Use nickel-NTA affinity chromatography for His-tagged protein

  • Follow with size exclusion chromatography for higher purity

  • Assess purity by SDS-PAGE (purified NME1 appears at approximately 19 kDa)

This methodology yields pure, functional NME1 protein suitable for biochemical, structural, and functional studies.

What experimental approaches can be used to study NME1's role in transcriptional regulation?

To investigate NME1's function as a transcriptional regulator, researchers can employ the following experimental approaches:

• Gene expression analysis:

  • qRT-PCR to measure target gene mRNA levels in response to NME1 manipulation

  • Analysis of pre-mRNA levels to distinguish transcriptional from post-transcriptional effects

• Promoter activity assays:

  • Construct promoter-luciferase reporter systems containing the promoter region of potential target genes

  • Measure luciferase activity in response to NME1 overexpression or knockdown

• Chromatin immunoprecipitation (ChIP):

  • Use anti-NME1 antibodies to immunoprecipitate NME1-bound chromatin

  • Identify NME1 binding sites at target gene promoters

  • Perform sequential ChIP to analyze co-occupancy with other transcription factors

• Epigenetic marker analysis:

  • Conduct ChIP for histone modifications (e.g., H3K4me3, H3K27ac) to assess changes in response to NME1

  • ChIP for RNA polymerase II recruitment to determine transcriptional activation

• Gene manipulation approaches:

  • Stable overexpression using plasmid transfection (e.g., pEGFP-N1-NME1)

  • Lentiviral transduction (e.g., pSMPUW-IRES-EGFP-NME1)

  • RNA interference or CRISPR-Cas9 for knockdown/knockout studies

These methodologies provide a comprehensive toolkit for examining NME1's direct and indirect effects on gene transcription.

How can researchers effectively study NME1's protein-protein interactions?

To investigate NME1's protein-protein interactions, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-NME1 antibodies to pull down NME1 along with interacting proteins

  • Identify binding partners through mass spectrometry

  • Confirm specific interactions with Western blotting

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to NME1

  • These methods are particularly useful for detecting transient or weak interactions

• Yeast two-hybrid screening:

  • Use NME1 as bait to screen cDNA libraries for potential interacting partners

  • Validate positive hits with alternative methods

• Fluorescence resonance energy transfer (FRET):

  • Tag NME1 and potential binding partners with appropriate fluorophores

  • Measure energy transfer as an indication of protein proximity in live cells

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC):

  • Determine binding kinetics and thermodynamics of purified proteins

  • Provide quantitative measures of binding affinity

These approaches can help elucidate NME1's complex interactome and provide insights into its diverse cellular functions.

How should researchers interpret contradictory findings regarding NME1's prognostic value in different cancer types?

The contradictory findings regarding NME1's prognostic value require careful interpretation based on multiple considerations:

• Tissue-specific context:

  • Different cellular environments may alter NME1's function

  • Analyze the tissue-specific signaling pathways that interact with NME1

  • Consider that NME1 may have opposing outcomes in different contexts, similar to observations with PI3K signaling

• Methodological differences:

  • Assess differences in detection methods (IHC, RT-PCR, Western blot)

  • Consider antibody specificity issues that may affect results

  • Evaluate whether studies distinguished between NME1 and other NME family members

• Molecular context analysis:

  • Examine co-expression of other genes that might modulate NME1 function

  • Consider post-translational modifications of NME1 in different tumor types

  • Evaluate the mutational status of NME1 (e.g., S120G mutation in neuroblastoma)

• Integrated biomarker approach:

  • Combine NME1 expression data with other molecular markers

  • Develop tissue-specific prognostic models that account for these variations

Researchers should avoid generalizing findings across cancer types and instead develop cancer-specific models for NME1's prognostic value.

What are the key considerations when analyzing NME1's role in complex signaling networks?

When investigating NME1's role in complex signaling networks, researchers should consider:

• Protein complexes and signaling hubs:

  • NME1 functions within larger protein complexes (e.g., with CFTR)

  • Study NME1 as part of signaling hubs rather than in isolation

  • Consider that disruption of these complexes (e.g., through CFTR mutation) may alter NME1 function

• Context-dependent signaling outcomes:

  • Similar to PI3K signaling, NME1 pathways may have opposing outcomes in different contexts

  • Examine tissue-specific and disease-specific signaling networks

• Integration of multiple functions:

  • Account for NME1's various functions (NDPK activity, transcriptional regulation, etc.)

  • Consider how these functions might interact or compete in specific cellular contexts

• Redox regulation:

  • NME1 undergoes various redox modifications including sulfonylation, CoAlation, glutathionylation, and intramolecular disulfide bond formation

  • These modifications may alter NME1's function in different cellular redox environments

• Experimental design considerations:

  • Use multiple model systems to validate findings

  • Employ both gain-of-function and loss-of-function approaches

  • Consider temporal aspects of signaling networks

Understanding these complex interactions will provide a more comprehensive view of NME1's role in cellular signaling and its implications for cancer biology.

What are the latest findings regarding NME1's role as a moonlighting protein?

Recent research has expanded our understanding of NME1 as a moonlighting protein with multiple functions:

• Coenzyme A binding:

  • NME1 has been identified as a major CoA-binding protein

  • This interaction suggests a potential regulatory mechanism affecting NME1's various functions

  • The binding occurs at the nucleotide-binding site in a non-covalent manner

• Redox regulation:

  • NME1 undergoes various redox modifications that may regulate its function

  • These modifications include sulfonylation, CoAlation, glutathionylation, and intramolecular disulfide bond formation

  • Cysteine residues C4, C109, and C145 are implicated in these redox-dependent regulations

• Direct transcriptional activation:

  • NME1 directly binds to promoter regions and enhances transcription

  • It recruits epigenetic activation markers and RNA polymerase II

  • This function expands NME1's role beyond its enzymatic NDPK activity

These emerging functions provide new perspectives on how NME1 regulates various cellular processes and how its dysregulation contributes to cancer progression.

What novel therapeutic approaches might target NME1 function in cancer?

Based on current understanding of NME1 biology, several therapeutic strategies could be developed:

• Restoring NME1 expression in metastatic cancers:

  • Epigenetic modifiers to reverse NME1 silencing

  • Targeted gene therapy approaches to deliver functional NME1

  • Small molecules that stabilize NME1 protein or enhance its activity

• Targeting NME1's protein-protein interactions:

  • Disrupting interactions with pro-metastatic binding partners

  • Enhancing interactions with anti-metastatic pathways

  • Developing peptidomimetics based on critical interaction domains

• Modulating NME1's transcriptional activity:

  • Enhancing NME1 binding to anti-metastatic gene promoters

  • Developing compounds that mimic NME1's effect on epigenetic markers

  • Targeting downstream effectors in the NME1 transcriptional network

• Exploiting redox regulation:

  • Developing compounds that modulate NME1's redox state

  • Targeting specific cysteine residues involved in redox regulation

  • Manipulating CoA binding to alter NME1 function

These approaches represent potential avenues for translating NME1 research into clinical applications, particularly for cancers where NME1 expression correlates with better prognosis.