NT5M Human

What is NT5M and what is its primary cellular function?

NT5M, also known as 5',3'-nucleotidase mitochondrial, deoxy-5'-nucleotidase 2 (dNT-2), or dNT2, is an enzyme that localizes to the mitochondrial matrix. Its primary function is dephosphorylating the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides, thereby protecting mitochondrial DNA replication from excess dTTP . The mature protein consists of 196 amino acids, formed after removal of the first 32 amino acids containing the mitochondrial targeting sequence . The NT5M gene is located on chromosome 17p11.2 in the critical region deleted in Smith-Magenis syndrome .

NT5M's enzymatic activity is highly specific, showing only marginal activity towards dIMP and dGMP . The enzyme forms part of a network of proteins involved in maintaining nucleotide balance within mitochondria, which is essential for proper mitochondrial DNA replication and transcription.

What is the structural organization of the NT5M enzyme?

The NT5M enzyme has a complex structure that determines its specific function. The enzyme likely functions as a dimer, with each monomer containing a large and small domain connected by two loops . The large domain forms an alpha/beta Rossmann fold with two helix loops, while the small domain forms a truncated four-helix bundle .

The active site is located in a cleft between the two domains and binds a magnesium ion that is coordinated by three exogenous ligands, a phosphate ion, and two water molecules in an octahedral arrangement . This structural configuration is crucial for its ability to recognize and dephosphorylate specific nucleotides.

What are the recommended methods for producing recombinant NT5M for in vitro studies?

For researchers studying NT5M in vitro, recombinant protein production is a critical starting point. NT5M Human Recombinant is typically produced in E. coli as a single, non-glycosylated polypeptide chain containing 218 amino acids (residues 32-228) with a molecular mass of approximately 25.1 kDa . The protein is usually fused to a 21 amino acid His-tag at the N-terminus to facilitate purification .

The recommended purification protocol involves:

• Expression in E. coli with appropriate induction parameters

• Cell lysis under conditions that preserve protein activity

• Purification using proprietary chromatographic techniques

• Formulation in a stabilizing buffer containing 20mM Tris-HCl (pH8.0), 20% glycerol, 0.1M NaCl, and 1mM DTT

For stability, store the purified protein at 4°C if using within 2-4 weeks, or at -20°C for longer storage periods . Adding a carrier protein (0.1% HSA or BSA) is recommended for long-term storage, and multiple freeze-thaw cycles should be avoided .

How can NT5M enzymatic activity be accurately measured?

Measuring NT5M activity requires sensitive methods that can differentiate its activity from other nucleotidases. Several approaches have been developed:

• Real-time PCR-based nucleotide quantification: This method can detect changes in specific dNTP pools resulting from NT5M activity. A reaction mixture containing Q5 reaction buffer, primers, template, limited dNTPs, and a fluorescent dye like EvaGreen can be used to quantify specific nucleotides via real-time PCR .

• Radioactive substrate assays: Using radiolabeled substrates (like [³H]-dTMP) followed by thin-layer chromatography or HPLC separation to quantify product formation.

• Fluorescent substrate analogs: Employing fluorescently labeled nucleotides whose dephosphorylation results in measurable changes in fluorescence.

• Compartment-specific analyses: Isolating mitochondria using differential centrifugation before performing activity assays to specifically measure mitochondrial NT5M activity.

For optimal results, researchers should consider:

• Using specific inhibitors to differentiate NT5M activity from other nucleotidases

• Conducting assays under conditions that favor NT5M activity (optimal pH and Mg²⁺ concentration)

• Including appropriate controls to account for background phosphatase activity

How does NT5M interact with other proteins in nucleotide metabolism pathways?

NT5M functions within a network of proteins involved in nucleotide metabolism. According to STRING interaction network data, NT5M has strong predicted functional partnerships with several key proteins :

These interactions suggest NT5M works in concert with other enzymes to maintain balanced nucleotide pools, particularly in mitochondria. Disruptions in these interactions may contribute to nucleotide imbalances and mitochondrial dysfunction.

To investigate these interactions experimentally, researchers can use:

• Co-immunoprecipitation followed by mass spectrometry

• Proximity-based labeling methods like BioID or APEX

• Fluorescence or bioluminescence resonance energy transfer (FRET/BRET)

• Protein fragment complementation assays

• Surface plasmon resonance to measure binding kinetics

What is the role of NT5M in mitochondrial DNA maintenance?

NT5M plays a crucial role in maintaining mitochondrial DNA (mtDNA) integrity by regulating nucleotide balance within the mitochondrial matrix. By dephosphorylating specific deoxyribonucleotides, NT5M helps prevent imbalances that could lead to mtDNA replication errors or depletion .

In non-replicating cells, where cytosolic dNTP synthesis is down-regulated, mtDNA synthesis depends heavily on mitochondrial enzymes like deoxyguanosine kinase (DGUOK) and thymidine kinase 2 (TK2) . NT5M works in concert with these enzymes to maintain appropriate nucleotide pools.

Experimental approaches to study NT5M's role in mtDNA maintenance include:

• Measuring mtDNA copy number in cells with modulated NT5M expression

• Assessing mtDNA mutation rates in NT5M-deficient models

• Monitoring mitochondrial nucleotide pools in response to NT5M manipulation

• Analyzing mitochondrial genome stability under conditions of nucleotide stress

• Investigating the effects of NT5M variants on mtDNA replication fidelity

How is NT5M being investigated in cancer research?

NT5M has emerged as a significant factor in cancer metabolism research. Studies have shown that modulating nucleotide metabolism through NT5M can affect cancer cell survival and proliferation :

• Nucleotide imbalance approach: Research has demonstrated that sustained consumption of dTMP (through expression of thymidylate 5'-phosphohydrolase) can induce dNTP imbalance in cancer cells, leading to apoptosis as tricarboxylic acid cycle intermediates become depleted .

• Metabolic vulnerability exploitation: This approach has shown particular promise in triple-negative breast cancer (TNBC) cell lines, where metabolic dependencies can be exploited to exacerbate cell metabolic vulnerability .

• mRNA-based therapies: Optimized mRNA expression platforms have been developed for delivering enzymes that can modify nucleotide balance in cancer cells . These platforms can potentially be used to express modified versions of NT5M or related enzymes.

The impact of NT5M modulation on cancer cell metabolism can be assessed through:

• Extracellular acidification rate (ECAR) analysis

• Oxygen consumption rate (OCR) measurement

• Differential transcription/expression profiling

• Metabolic flux analysis using labeled substrates

• Cell viability and apoptosis assays under various nutrient conditions

What is known about NT5M in relation to genetic disorders?

NT5M's location within the Smith-Magenis syndrome (SMS) critical region on chromosome 17p11.2 suggests potential involvement in this genetic disorder . While the specific contribution of NT5M to SMS phenotypes remains under investigation, several lines of evidence connect NT5M to genetic disorders:

• Smith-Magenis syndrome: This neurodevelopmental disorder is caused by a deletion in chromosome 17p11.2 that includes the NT5M gene . The role of NT5M in contributing to SMS phenotypes remains an active area of research.

• Mitochondrial DNA depletion syndromes: Given NT5M's role in mitochondrial nucleotide metabolism, variants affecting its function may contribute to mtDNA depletion syndromes, particularly in tissues with high energy demands.

• Chemical sensitivity: Studies in rodent models have shown that NT5M expression is affected by various chemicals including tetrachlorodibenzodioxine, 5-fluorouracil, and all-trans-retinoic acid, suggesting a role in toxicological response pathways .

Research methodologies in this area include:

• Genotype-phenotype correlation studies in patient cohorts

• Functional characterization of NT5M variants identified in patients

• Animal models with targeted NT5M modifications

• Cell-based assays measuring mitochondrial function in patient-derived cells

How do the substrate specificities of NT5M compare to other nucleotidases?

NT5M has distinct substrate specificities that differentiate it from other nucleotidases. It specifically dephosphorylates the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides, with only marginal activity towards dIMP and dGMP . This specificity is crucial for its role in mitochondrial nucleotide metabolism.

Among the seven 5'-nucleotidases identified in humans, NT5M's closest relative is the cytosolic 5'-nucleotidase (cdN), with which it shares 52% amino acid sequence identity . Their genes, NT5M and NT5C, share the same exon/intron organization .

Researchers can differentiate between these enzymes using:

• Subcellular fractionation to isolate different cellular compartments

• Substrate panels to identify signature substrate preference patterns

• Specific inhibitors that differentially affect various nucleotidases

• Analysis of optimal reaction conditions (pH, metal ion requirements)

What are the cutting-edge research questions surrounding NT5M regulation?

Several unresolved questions about NT5M regulation present exciting opportunities for novel research:

• Post-translational modifications: How do phosphorylation, acetylation, or other modifications regulate NT5M activity in response to changing cellular conditions? Recent advances in proteomics enable comprehensive mapping of such modifications.

• Metabolic sensing: Does NT5M activity respond to cellular energy status or redox state? This question could be addressed using metabolic stress models and activity assays under varied conditions.

• Transcriptional regulation: What factors control NT5M expression in different tissues and under different physiological states? Chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis approaches can help identify regulatory elements.

• Mitochondrial import regulation: How is the transport of NT5M into mitochondria regulated, and does this represent a control point for its activity? In vitro import assays and imaging approaches can address this question.

• Protein-protein interactions: What protein complexes does NT5M form within mitochondria, and how do these interactions modulate its function? Advanced proteomics approaches like BioID or APEX2 proximity labeling can identify interaction partners.

• Allosteric regulation: Are there allosteric regulators of NT5M activity that respond to mitochondrial nucleotide levels? Structural studies combined with enzymatic assays could reveal such mechanisms.

These research directions require cutting-edge technologies like CRISPR-based genome editing, advanced imaging techniques, metabolomics, and computational modeling to fully elucidate NT5M's regulatory networks.

What innovative therapeutic approaches might target NT5M?

Emerging research suggests several innovative therapeutic approaches targeting NT5M or leveraging its function:

• Cancer metabolism targeting: Since modulation of NT5M activity can induce nucleotide imbalances particularly detrimental to metabolically vulnerable cancer cells, therapeutic strategies might include:

  • Small molecule inhibitors of NT5M based on its crystal structure

  • mRNA-based approaches to express modified nucleotidases in cancer cells

  • Combination therapies targeting NT5M alongside conventional chemotherapeutics

• Mitochondrial disorder treatments: For conditions involving mitochondrial nucleotide imbalance, approaches might include:

  • Gene therapy to correct NT5M deficiencies

  • Small molecules that modulate NT5M activity

  • Nucleotide supplementation strategies to bypass NT5M-related defects

• Drug resistance mechanisms: NT5M might be involved in resistance to nucleoside analog drugs used in cancer, viral infections, or bacterial diseases. Understanding these mechanisms could lead to:

  • Combination therapies that overcome resistance

  • Biomarkers predicting drug response based on NT5M status

  • Novel drug designs that bypass NT5M-mediated resistance

The development of these approaches requires:

• High-throughput screening platforms for NT5M modulators

• Patient-derived organoid or xenograft models for testing efficacy

• Pharmacokinetic and pharmacodynamic studies of candidate compounds

• Biomarker development to identify patients likely to respond to NT5M-targeted therapies

How might emerging technologies advance NT5M research?

Several cutting-edge technologies are poised to significantly advance our understanding of NT5M:

• CRISPR-based technologies:

  • Base editors and prime editors allow precise introduction of NT5M variants without double-strand breaks

  • CRISPRi/CRISPRa systems enable reversible modulation of NT5M expression

  • CRISPR screens can identify synthetic lethal interactions with NT5M in different contexts

• Advanced imaging technologies:

  • Super-resolution microscopy can visualize NT5M distribution within mitochondrial subcompartments

  • Live-cell imaging with genetically encoded biosensors can track nucleotide dynamics in real-time

  • Correlative light and electron microscopy can connect NT5M localization to ultrastructural features

• Single-cell and spatial technologies:

  • Single-cell transcriptomics and proteomics can identify cell populations with distinct NT5M expression patterns

  • Spatial transcriptomics can map NT5M expression in tissue contexts

  • Single-cell metabolomics might detect cell-to-cell variation in nucleotide metabolism

• Structural biology advances:

  • Cryo-electron microscopy to visualize NT5M in complex with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • AI-based structural prediction tools like AlphaFold to model NT5M complexes and interactions

• Metabolic analysis tools:

  • Real-time metabolite sensing using genetically encoded fluorescent sensors

  • Stable isotope-resolved metabolomics to track nucleotide flux

  • Computational modeling integrating multi-omics data

These technologies collectively provide unprecedented resolution in understanding NT5M's dynamic role in mitochondrial function and cellular metabolism, potentially leading to novel therapeutic strategies for related disorders.