NME5 Antibody

What is NME5 and what are its primary biological functions?

NME5 (non-metastatic cells 5, protein expressed in nucleoside-diphosphate kinase) is a member of the NME/NM23 gene family. Despite containing a conserved nucleoside diphosphate kinase domain, human NME5 does not appear to possess NDK kinase activity .

The protein functions primarily through:

• Protection against cell death induced by Bax

• Modulation of cellular antioxidant enzyme levels, including Gpx5

• Supporting spermiogenesis by enhancing reactive oxygen species elimination in late-stage spermatids

• Functioning as a component of axonemal radial spoke complexes critical for sperm and cilia motility

• Exhibiting 3'-5' exonuclease activity with preference for single-stranded DNA, suggesting roles in DNA proofreading and repair

Evolutionarily, it's noteworthy that ancestral Nme5-like proteins (such as those found in red algae like Chondrus crispus) retained full nucleoside diphosphate kinase activity, unlike the human homolog which has lost this function .

How is NME5 protein expression distributed across tissue types?

NME5 shows distinct tissue expression patterns with highest expression observed in:

In ciliated epithelium, immunogold electron microscopy has shown that NME5 localizes primarily to ciliary structures, with the most frequent binding sites in the region of inner dynein arms near the outer microtubules . This localization pattern is consistent with its proposed role in ciliary function and motility.

What criteria should researchers consider when selecting an NME5 antibody?

When selecting an NME5 antibody, researchers should consider multiple parameters to ensure experimental success:

For critical experiments, select antibodies with multiple validation methods and published literature support. Most commercially available NME5 antibodies are rabbit polyclonals with reactivity against human and mouse NME5 .

How should researchers validate the specificity of NME5 antibodies?

A comprehensive validation approach should include:

• Positive and negative controls:

  • Use cell lines with known NME5 expression (A431, HeLa, NIH-3T3 as positive controls)

  • Compare with tissues from knockout models when available (as demonstrated in PCD-affected Alaskan Malamutes)

• Complementary techniques:

  • Western blot to confirm molecular weight (expected ~24 kDa)

  • Immunofluorescence to verify subcellular localization patterns

  • RNA expression data (RT-PCR or RNAseq) to correlate with protein detection

• Functional validation:

  • Knockdown studies with siRNA to demonstrate specificity

  • Appropriate peptide blocking controls

Evidence of successful validation has been demonstrated in studies of NME5 in PCD, where immunohistochemistry confirmed absence of NME5 protein expression in nasal epithelium of affected dogs compared to controls .

What are the optimal conditions for using NME5 antibodies in immunohistochemistry (IHC)?

Optimal IHC protocols for NME5 detection should consider:

• Sample preparation:

  • PFA fixation is suitable for most applications

  • Both paraffin-embedded and frozen sections can be used

• Antibody dilutions:

  • Recommended range: 1:20-1:2500 (optimize for each antibody)

  • Typical working dilutions: 1:1000-1:2500 for most commercial antibodies

• Detection systems:

  • DAB-based chromogenic detection works well for tissue localization

  • Fluorescent detection may provide better resolution for subcellular localization

• Controls:

  • Include testis tissue as positive control

  • Primary antibody omission as negative control

Studies examining NME5 in ciliated epithelium have successfully employed these approaches to demonstrate specific localization patterns in both normal and disease states .

What protocols are recommended for Western blot analysis of NME5?

For optimal Western blot detection of NME5:

• Protein extraction:

  • Standard RIPA buffer is suitable for most applications

  • For ciliated tissues, consider specialized extraction buffers to ensure solubilization of axonemal proteins

• Running conditions:

  • 10-12% SDS-PAGE gels

  • Expected molecular weight: ~24 kDa

• Transfer and blocking:

  • PVDF membranes work well

  • 5% non-fat dry milk or BSA in TBST for blocking

• Antibody incubation:

  • Primary: 1:500-1:2000 dilution range

  • Typically overnight at 4°C

• Detection system:

  • Enhanced chemiluminescence (ECL) provides sufficient sensitivity

  • For lower expression tissues, consider more sensitive detection methods

When analyzing NME5 multimeric structures (as seen in ancestral forms like Nme5-likeCc), non-denaturing gel conditions may be required to preserve oligomeric states .

How does NME5 contribute to gemcitabine resistance in pancreatic cancer?

NME5 has been identified as a contributor to innate gemcitabine resistance in pancreatic cancer through several mechanisms:

• Apoptosis modulation:

  • NME5 attenuates apoptosis induction by gemcitabine

  • Downregulation of NME5 significantly reverses gemcitabine resistance in resistant cell lines (PAXC002)

• Cell cycle regulation:

  • NME5 blunts gemcitabine-induced cell cycle arrest

  • Acts as a functional link between drug response and cell cycle progression

• NF-κB pathway interaction:

  • NME5 directly binds to NF-κB

  • Regulates NF-κB expression levels in resistant cells

  • NF-κB functions as a key executor of NME5 in regulating apoptosis and cell cycle

Experimentally, researchers have demonstrated that:

• NME5 knockdown significantly reverses gemcitabine resistance

• NME5 overexpression induces gemcitabine resistance in previously sensitive cell lines

• These effects operate through NF-κB-dependent mechanisms

These findings suggest NME5 as a potential therapeutic target for overcoming gemcitabine resistance in pancreatic cancer treatment.

What is the role of NME5 in primary ciliary dyskinesia and other ciliopathies?

NME5 plays a critical role in cilia function, as evidenced by its involvement in primary ciliary dyskinesia (PCD):

• Genetic evidence:

  • A frameshift variant in the NME5 gene (c.43delA) has been identified in Alaskan Malamutes with PCD

  • This mutation leads to an early frame-shift and premature stop codon

  • Perfect concordance between NME5 genotypes and PCD phenotypes was observed in affected dogs

• Structural impact:

  • NME5 is a component of ciliary structures, particularly associated with radial spokes

  • Immunogold TEM localized NME5 to the region of inner dynein arms near outer microtubules

  • Loss of NME5 expression leads to defects in outer and particularly inner dynein arms

• Functional confirmation:

  • Immunohistochemistry confirmed absence of NME5 protein expression in nasal epithelium of affected dogs

  • Electron microscopy showed altered binding patterns in affected tissues

This research identifies NME5 as a candidate gene for human PCD cases and demonstrates its essential role in maintaining proper ciliary structure and function.

How has the evolutionary structure-function relationship of NME5 changed across species?

NME5 has undergone significant evolutionary changes that affect its structure and function:

• Ancestral vs. human NME5:

  • Ancestral Nme5-like protein (as found in red algae Chondrus crispus) was a fully functional multimeric NDP kinase

  • Human NME5 has lost this enzymatic activity

• Structural differences:

  • Two amino acid deletion in the α1 helix of human NME5

  • Three amino acid insertion in the Kpn-loop

  • These changes cause significant structural alterations that likely prevent hexamer assembly and/or enzymatic activity

• Functional implications:

  • Ancestral NME5 forms high-molecular (>250 kDa) oligomeric structures

  • Human NME5 has evolved different functions related to apoptosis regulation and antioxidant response

  • The ATP binding cavity is better conserved in ancestral forms than in human NME5

This evolutionary divergence suggests functional specialization of NME5 in higher organisms, possibly related to its roles in specialized ciliated cells and apoptosis regulation.

What methodological approaches are recommended for studying NME5 protein interactions?

To investigate NME5 protein interactions, researchers should consider:

• Co-immunoprecipitation:

  • Successfully used to demonstrate direct binding between NME5 and NF-κB

  • Requires optimization of antibody concentrations and binding conditions

  • Recommended dilution for IP applications: refer to manufacturer's guidelines (typically 1:50-1:200)

• Proximity ligation assays:

  • Useful for detecting protein-protein interactions in situ

  • Can provide spatial context for NME5 interactions within ciliary structures

• Recombinant protein studies:

  • Expression and purification of NME5 can be used for direct binding assays

  • Gel filtration and cross-linking with glutaraldehyde have been used successfully to study oligomeric states

• Functional validation:

  • Knockdown/overexpression approaches followed by assessment of interacting partner function

  • Analysis of downstream pathway activity (e.g., NF-κB pathway activation)

These approaches have successfully revealed NME5's interaction with NF-κB in gemcitabine resistance studies and can be adapted to investigate other potential binding partners .

What are the most common technical challenges when working with NME5 antibodies?

Researchers frequently encounter these challenges when working with NME5 antibodies:

• Tissue-specific expression levels:

  • NME5 shows highest expression in testis with lower levels in other tissues

  • May require more sensitive detection methods for low-expressing tissues

  • Consider concentration steps or signal amplification systems

• Specificity concerns:

  • Cross-reactivity with other NME family members is possible

  • Validate specificity using knockout/knockdown controls

  • Consider epitope-specific antibodies targeting unique regions of NME5

• Antibody recycling limitations:

  • Not recommended due to changes in buffer system after use

  • If necessary, high-titer antibodies can be reused approximately three times

  • Store recycled antibodies at 4°C for no more than one week

• Fixation sensitivity:

  • Some epitopes may be sensitive to particular fixation methods

  • Compare multiple fixation protocols if initial attempts fail

  • PFA fixation is generally suitable for most applications

When encountering difficulties, comparing results from multiple antibodies targeting different epitopes of NME5 can help ensure reliable detection.

How should researchers interpret contradictory results between different NME5 antibodies?

When facing contradictory results from different NME5 antibodies:

• Evaluate antibody characteristics:

  • Compare immunogen sequences to identify potential epitope differences

  • Different epitopes may be differentially accessible in certain contexts

  • Check if antibodies recognize distinct domains or isoforms

• Consider technical factors:

  • Application-specific performance (an antibody optimized for WB may perform poorly in IHC)

  • Dilution optimization may be needed for each application

  • Buffer conditions can affect epitope recognition

• Validation approaches:

  • Use orthogonal methods to confirm results (e.g., mRNA expression)

  • Employ genetic models (knockdown/knockout) when available

  • Consider peptide competition assays to confirm specificity

• Biological explanations:

  • Post-translational modifications may affect epitope recognition

  • Protein interactions might mask certain epitopes

  • Subcellular localization differences could explain discrepancies

What are the promising areas for future NME5 research based on current knowledge?

Several promising research directions merit further investigation:

• Cancer therapy resistance mechanisms:

  • Expand studies of NME5's role in chemoresistance beyond pancreatic cancer

  • Investigate potential as a therapeutic target or biomarker

  • Explore combinations of NME5 inhibition with standard therapies

• Ciliopathy connections:

  • Screen for NME5 mutations in human PCD cases without known genetic causes

  • Develop animal models to further elucidate ciliary functions

  • Investigate tissue-specific effects of NME5 dysregulation

• Structural biology approaches:

  • Resolve crystal structure of human NME5 to understand functional divergence

  • Compare with ancestral forms to identify key evolutionary changes

  • Design structure-based modulators of NME5 function

• Oxidative stress response mechanisms:

  • Further explore the role of NME5 in antioxidant enzyme regulation

  • Investigate potential therapeutic applications in conditions with oxidative stress

  • Study the specific molecular pathways connecting NME5 to antioxidant systems

These areas represent critical knowledge gaps that, when addressed, could significantly advance our understanding of NME5 biology and its applications in human disease.

What methodological innovations might improve NME5 research?

Emerging technologies that could enhance NME5 research include:

• CRISPR/Cas9 gene editing:

  • Creation of precise NME5 knockout or knock-in models

  • Introduction of disease-specific mutations

  • Development of reporter systems for studying NME5 regulation

• Advanced imaging approaches:

  • Super-resolution microscopy for detailed ciliary localization

  • Live-cell imaging to track NME5 dynamics

  • Cryo-EM for structural studies of NME5 complexes

• Single-cell analyses:

  • Single-cell proteomics to detect cell-type specific expression patterns

  • Combined transcriptome/proteome approaches to correlate expression levels

  • Spatial transcriptomics to map tissue distribution with higher precision

• Computational approaches:

  • AI-driven prediction of NME5 interactions and functional networks

  • Molecular dynamics simulations to understand structural characteristics

  • Systems biology approaches to position NME5 within cellular pathways