NME3 Antibody

What is NME3 and what are its primary biological functions?

NME3 (NME/NM23 nucleoside diphosphate kinase 3) is a member of the nucleoside diphosphate kinase family that plays critical roles in multiple cellular processes. Its primary biochemical function involves catalyzing the phosphorylation of ribonucleosides and deoxyribonucleoside diphosphates (except ATP) into corresponding triphosphates, with ATP serving as the major phosphate donor .

The ATP gamma phosphate is transferred to the nucleoside diphosphate beta phosphate via a ping-pong mechanism using a phosphorylated active-site intermediate . This catalytic activity contributes to the regulation of intracellular nucleotide homeostasis.

Beyond its enzymatic function, NME3 has several other biological roles:

• Facilitates mitochondrial tethering and fusion through membrane binding and hexamerization

• Plays a role in DNA repair mechanisms for both single and double-stranded breaks

• May be required for ciliary function during renal development

• Inhibits granulocyte differentiation in hematopoiesis

• Functions as a gatekeeper for DRP1-dependent mitophagy during hypoxic conditions

Where is NME3 localized in cells and how can researchers verify its subcellular distribution?

NME3 primarily localizes to the mitochondrial outer membrane, with this localization dependent on its N-terminal region (first 30 amino acids) . Researchers have demonstrated this localization through several methodological approaches:

Methodology for verifying NME3 localization:

• Confocal microscopy: Immunofluorescence staining reveals colocalization of NME3 with mitochondrial markers like COX4

• Super-resolution microscopy: Reveals more precise localization at the mitochondrial outer membrane, showing costaining patterns with TOMM20, an outer membrane protein

• Proteinase K digestion assay: NME3, like other outer membrane proteins (MFN1, MFN2, TOMM20), is susceptible to proteinase K digestion, while matrix proteins remain protected unless membranes are disrupted

• Subcellular fractionation: Isolation of mitochondria followed by immunoblotting can confirm mitochondrial localization

• Deletion mutant analysis: The N-terminal 30-amino acid-deleted (ΔN) NME3 mutant fails to localize to mitochondria, demonstrating the necessity of this region for proper localization

Interestingly, while primarily localized to mitochondria, NME3 has also been found to interact with nephronophthisis-related proteins (NPHPs) and can be recruited to DNA damage sites, suggesting context-dependent localization .

What applications are NME3 antibodies commonly used for in research?

NME3 antibodies are valuable tools for investigating various aspects of NME3 biology. Based on antibody validation data, their applications include:

When selecting an NME3 antibody, researchers should consider:

• Target epitope (N-terminal, middle region, C-terminal)

• Validated applications and species reactivity

• Clonality (monoclonal versus polyclonal)

• Detection method (direct versus indirect)

How does NME3 contribute to mitochondrial dynamics and what experimental approaches can verify this function?

NME3 plays a critical role in mitochondrial dynamics through both kinase-dependent and kinase-independent mechanisms. Research has shown that:

• NME3 interacts with mitofusins: NME3 forms complexes with MFN1 and MFN2, key GTPases that mediate mitochondrial outer membrane fusion

• Loss of NME3 impacts mitochondrial fusion: NME3 knockdown or depletion results in slower mitochondrial dynamics and fusion rates

• Hexamerization function is crucial: NME3's ability to oligomerize appears important for facilitating mitochondrial fusion, independent of its kinase activity

Experimental approaches to verify NME3's role in mitochondrial dynamics:

• Photoactivatable-GFP (PA-GFP) mitochondrial fusion assay:

  • Methodology: Express mitochondrially-targeted PA-GFP, photoactivate a subset of mitochondria, and track the spread of fluorescence over time

  • Expected results: NME3 knockdown cells show slower fluorescence spread compared to control cells, indicating reduced fusion rates

• Proximity ligation assay (PLA):

  • Methodology: Use antibodies against NME3 and mitochondrial fusion proteins (MFN1/2) to detect in situ protein interactions

  • Expected results: PLA signals between NME3 and MFN1/2 are detected, while little signal is observed between NME3 and other mitochondrial proteins like TOMM20 or DRP1

• Rescue experiments with domain mutants:

  • Methodology: Express wild-type NME3, catalytic-dead (H135Q), or oligomerization-deficient (E40D/E46D) mutants in NME3-deficient cells

  • Expected results: Wild-type and catalytic-dead NME3 restore mitochondrial fusion, while oligomerization-deficient mutants fail to rescue the phenotype

• Mitochondrial morphology analysis during stress:

  • Methodology: Subject cells to stresses like glucose starvation or hypoxia and analyze mitochondrial network morphology

  • Expected results: NME3-deficient cells fail to undergo stress-induced mitochondrial elongation that normally serves as a protective response

What is the relationship between NME3 and hypoxia-induced mitophagy, and how can researchers investigate this process?

Recent research has revealed that NME3 functions as a critical gatekeeper for DRP1-dependent mitophagy during hypoxia . This process depends on NME3's active site phosphohistidine (H135) but is independent of its nucleoside diphosphate kinase function.

Key findings on NME3's role in hypoxia-induced mitophagy:

• NME3 is required for hypoxia-induced mitophagy as demonstrated by reduced mitophagy in NME3 knockdown cells exposed to hypoxia

• This function depends on the catalytic histidine 135 site but is independent of NME3's NDPK function

• Hypoxia treatment increases DRP1 interaction with NME3 in a phosphohistidine-dependent manner

• MUL1, a ubiquitin E3 ligase, is involved in the reduction of active DRP1 and impairment of hypoxia-induced mitophagy in NME3-deficient cells

Methodological approaches to study NME3 in hypoxia-induced mitophagy:

• Mt-Keima assay for mitophagy quantification:

  • Methodology: Express the dual-excitation mitochondrial-targeted Keima fluorescent protein, which changes fluorescence properties based on pH, allowing distinction between mitochondria in neutral cytosol versus acidic lysosomes

  • Analysis: Quantify red/green fluorescence ratio under normoxia versus hypoxia conditions in control and NME3-depleted cells

• Hypoxia chamber experiments:

  • Methodology: Incubate cells in 0.5% oxygen hypoxia chamber for 24 hours with or without lysosomal inhibitors like chloroquine

  • Readouts: Analyze LC3B puncta formation, mitochondrial fragmentation, and cell viability

• Protein interaction studies during hypoxia:

  • Methodology: Use super-resolution confocal microscopy and co-immunoprecipitation to assess NME3-DRP1 interaction

  • Finding: Hypoxia treatment stimulates interaction between endogenous NME3 and DRP1

• Mutant rescue experiments:

  • Methodology: Express shRNA-resistant wild-type (WT), H135Q (HQ), or E40/46D (ED) mutants in NME3-depleted cells and expose to hypoxia

  • Results: WT and ED mutants restore hypoxia-induced mitophagy while H135Q mutant fails to rescue

How does NME3 interact with nephronophthisis-related proteins, and what methods are optimal for studying these interactions?

NME3 has been identified as a binding partner for several nephronophthisis-related proteins (NPHPs), suggesting a potential role in renal ciliopathies . Understanding these interactions provides insight into both NME3 function and ciliopathy pathogenesis.

Key NME3-NPHP interactions:

• NME3 binds to NPHP3, NEK8, ANKS6, NPHP1, and NPHP4

• The N-terminal 17 amino acids of NME3 are essential for interaction with NEK8 and ANKS6

• NME3 may act as a link between different NPHP modules by facilitating binding between proteins that don't interact directly (e.g., NPHP1/NPHP4 and ANKS6)

Methodological approaches to study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Methodology: Immunoprecipitate NME3 or NPHPs (NPHP1, NPHP4, NEK8, ANKS6) from cell lysates and detect interacting partners by immunoblotting

  • Applications: Determine direct protein-protein interactions and map interaction domains

  • Example result: Immunoprecipitation of either NPHP1 or NPHP4 revealed that coexpression of NME3 facilitated binding to ANKS6

• Fragment analysis:

  • Methodology: Generate deletion constructs of NME3 and test their ability to interact with NPHPs

  • Finding: The N-terminal 17 amino acids of NME3 are essential for interaction with NEK8 and ANKS6

• Proximity ligation assay (PLA):

  • Methodology: Use antibodies against NME3 and various NPHPs to detect in situ protein interactions

  • Advantage: Allows visualization of protein complexes in their native cellular context

• Yeast two-hybrid assay:

  • Methodology: Test direct protein-protein interactions using NME3 and NPHPs as bait and prey

  • Application: Used to identify NPHP3 as an interaction partner for NEK6

• Immunofluorescence colocalization:

  • Methodology: Co-stain cells for NME3 and NPHPs and analyze using confocal microscopy

  • Expected results: Colocalization at basal bodies of primary cilia where interactions with NEK8 and CEP164 may take place

How can researchers distinguish between the nucleoside diphosphate kinase activity and the structural functions of NME3?

NME3 exhibits dual functionality: enzymatic activity as a nucleoside diphosphate kinase and structural roles independent of this catalytic function. Distinguishing between these functions is crucial for understanding NME3's diverse cellular roles.

Strategies to separate enzymatic and structural functions:

• Site-directed mutagenesis of functional domains:

  • Catalytic mutants: The H135Q mutation creates a catalytically inactive NME3 while preserving structural integrity

  • Oligomerization mutants: The E40D/E46D double mutation reduces hexamerization capacity while maintaining catalytic activity

  • Localization mutants: Deletion of the N-terminal 30 amino acids (ΔN) prevents mitochondrial localization

• Functional rescue experiments:

  Function	Wild-type NME3	H135Q (Catalytic Dead)	E40D/E46D (Oligomerization Deficient)
  Mitochondrial fusion	Rescues	Rescues	Fails to rescue
  Mitochondrial elongation during glucose starvation	Rescues	Rescues	Fails to rescue
  ATP production during glucose starvation	Rescues	Fails to rescue	Fails to rescue
  Hypoxia-induced mitophagy	Rescues	Fails to rescue	Rescues
  Cell viability under stress	Rescues	Fails to rescue	Fails to rescue

  This pattern of rescue demonstrates that:

  • Mitochondrial fusion requires NME3's structural properties but not its kinase activity

  • Cell survival under energy stress requires both NME3's structural properties and kinase activity

  • Hypoxia-induced mitophagy depends on the catalytic histidine but not the NDPK function

• In vitro enzyme activity assays:

  • Methodology: Purify recombinant wild-type and mutant NME3 proteins and measure nucleoside diphosphate kinase activity

  • Expected results: Wild-type NME3, but not H135Q mutant, catalyzes phosphate transfer from ATP to NDPs

• Subcellular context:

  • DNA damage sites: NME3 plays a role in DNA repair independent of its kinase activity through association with the ribonucleotide reductase complex via KAT5

  • Mitochondria: NME3 facilitates mitochondrial fusion through physical interaction with MFN1/2, independent of kinase activity

What genetic and cellular models are available to study NME3 function, and what phenotypes do they exhibit?

Various genetic and cellular models have been developed to investigate NME3 function in different contexts:

Cellular models:

• Knockdown/knockout cell lines:

  • NME3 siRNA or shRNA in human cell lines (HeLa, fibroblasts)

  • CRISPR-Cas9 engineered NME3 knockout cells

  • Phenotypes: Slower mitochondrial dynamics, impaired hypoxia-induced mitophagy, increased susceptibility to metabolic stress

• Patient-derived fibroblasts:

  • Fibroblasts from a patient (F741) with homozygous initiation-codon mutation in NME3

  • Phenotypes: NME3 protein deficiency, abnormal mitochondrial dynamics, abnormal cristae morphology, vulnerability to glucose starvation

• Expression systems for structure-function analysis:

  • Cells expressing wild-type or mutant NME3 (H135Q, E40D/E46D, ΔN)

  • Applications: Rescue experiments, localization studies, protein interaction analysis

• Endogenous tagging systems:

  • CRISPR-Cas9 mediated GFP or HA knock-in at NME3 C-terminus

  • Applications: Visualization of endogenous NME3 localization and dynamics

Animal models:

• NME3-deficient mice:

  • Phenotype: Congenital hypotonia and hypoventilation reported in patients with NME3 mutations

• NME3 H135Q knock-in mice:

  • Models the catalytically inactive form of NME3

  • Applications: Studying the importance of NME3 catalytic activity in vivo

• Mouse embryonic fibroblasts (MEFs):

  • Derived from NME3 wild-type and mutant mice

  • Applications: Ex vivo studies of NME3 function in primary cells

Experimental approaches using these models:

• Phenotypic analysis:

  • Mitochondrial network morphology (fragmented versus fused)

  • Response to cellular stresses (glucose starvation, hypoxia)

  • Cell viability and metabolic measurements

  • Mitophagy quantification

• Rescue experiments:

  • Re-expression of wild-type or mutant NME3 in deficient cells

  • Analysis of which functions are restored by which constructs

• Genetic screening:

  • Targeted sequencing of NME3 in ciliopathy patients (e.g., nephronophthisis)

  • Investigation of potential disease-causing mutations

What are the challenges in detecting endogenous NME3, and how can researchers overcome them?

Detecting endogenous NME3 presents several technical challenges that researchers should be aware of:

Common challenges:

• Limited antibody specificity: Some studies note difficulties in obtaining specific anti-NME3 antibodies from commercial sources or in-house preparation

• Low expression levels: Endogenous NME3 may be expressed at levels difficult to detect in some cell types

• Cross-reactivity with other NME family members: The NME family has multiple members with sequence similarity

• Context-dependent localization: NME3 localizes to different cellular compartments depending on context

Strategies to overcome these challenges:

• Antibody validation approaches:

  • Use NME3 knockdown or knockout cells as negative controls

  • Test multiple antibodies targeting different epitopes

  • Verify specificity by immunoprecipitation followed by mass spectrometry

  • Use phospho-specific antibodies when studying the active state of NME3

• Tagging strategies for endogenous NME3:

  • CRISPR-Cas9 mediated knock-in of small epitope tags (HA, Flag) or fluorescent proteins (GFP)

  • This approach allows detection of endogenous NME3 without overexpression artifacts

• Mass spectrometry-based detection:

  • For tissues or samples where antibody-based detection is problematic

  • Can provide quantitative information on NME3 expression levels

• Subcellular fractionation:

  • Concentrate NME3 by isolating mitochondrial fractions before detection

  • Enhances detection sensitivity when studying mitochondrial NME3

• mRNA detection as a proxy:

  • qRT-PCR for NME3 transcript levels

  • RNA-seq analysis for expression patterns across tissues or conditions

How can researchers effectively study the phosphohistidine-dependent functions of NME3?

The catalytic histidine residue (H135) of NME3 is critical for several of its functions. Studying phosphohistidine is technically challenging due to the instability of this modification, but several approaches can be employed:

Methodological approaches:

• Site-directed mutagenesis:

  • Generate H135Q mutant to abolish histidine phosphorylation

  • Compare functions of wild-type and H135Q NME3 to identify phosphohistidine-dependent processes

• Phosphohistidine-specific antibodies:

  • Use newly developed antibodies that specifically recognize phosphohistidine

  • Applications include immunoblotting, immunofluorescence, and immunoprecipitation

• Acid-labile phosphorylation detection:

  • Phosphohistidine is acid-labile unlike phosphoserine/threonine/tyrosine

  • Compare phosphoprotein staining after neutral versus acidic treatment

• 32P-labeling experiments:

  • Metabolic labeling with 32P followed by immunoprecipitation of NME3

  • Analyze phosphorylation status of wild-type versus mutant proteins

• Functional assays coupled with mutation analysis:

  • For hypoxia-induced mitophagy: Compare mt-Keima fluorescence in cells expressing wild-type versus H135Q NME3

  • For mitochondrial fusion: Analyze fusion rates in cells expressing different NME3 variants

• Mass spectrometry approaches:

  • Use neutral or basic conditions during sample preparation to preserve phosphohistidine

  • Targeted mass spectrometry methods can detect and quantify phosphohistidine

What considerations should be taken into account when designing experiments to study the role of NME3 in disease models?

When investigating NME3 in disease contexts, researchers should consider several experimental design factors:

Disease contexts associated with NME3:

• Ciliopathies: NME3 interacts with nephronophthisis-related proteins and may contribute to renal development

• Neurodegenerative disorders: A homozygous initiation-codon mutation in NME3 was identified in a patient with fatal infantile neurodegenerative disorder

• Hematopoietic disorders: NME3 plays a role in myeloid differentiation and could contribute to leukemia when dysregulated

• Mitochondrial diseases: NME3's role in mitochondrial dynamics suggests potential involvement in mitochondriopathies

Experimental design considerations:

• Model selection:

  • Cell line models: Choose disease-relevant cell types (renal cells for ciliopathies, neuronal cells for neurodegenerative disorders)

  • Patient-derived cells: When available, use cells from patients with suspected NME3-related disorders

  • Animal models: Consider tissue-specific conditional knockouts to avoid embryonic lethality

• Physiological relevance:

  • Study NME3 under stress conditions relevant to disease (hypoxia, metabolic stress)

  • Use physiological expression levels rather than overexpression when possible

  • Consider developmental timing when studying congenital disorders

• Functional readouts:

  • For ciliopathies: Ciliogenesis, ciliary length, and signaling pathway analysis

  • For neurodegenerative disorders: Mitochondrial function, ATP production, cell viability

  • For hematopoietic disorders: Differentiation assays, colony formation

  • For all contexts: Mitochondrial network morphology and dynamics

• Genetic approach considerations:

  • Use disease-specific mutations rather than complete knockouts when studying patient-associated variants

  • For hypomorphic mutations, titrate knockdown levels to match patient expression

  • Consider compensatory mechanisms that may mask phenotypes in complete knockout models

• Molecular mechanism investigation:

  • Distinguish between enzymatic and structural roles of NME3 in the disease context

  • Test rescue with specific domain mutants to identify critical functions

  • Consider interactions with disease-associated proteins

What are emerging areas of investigation regarding NME3 function and regulation?

Based on current research, several promising directions for future NME3 research include:

• Role in ciliary GTP homeostasis:

  • NME3 may provide GTP substrate for ciliary GTPases like RPGR, GPR48, RanGDP/GTP, ARL6, IFT22, and IFT27

  • Development of genetically encoded GTP sensors targeted to cilia could help determine whether ciliary GTP concentrations depend on NME3

• Mechanisms of NME3-mediated DNA repair:

  • Further investigation of NME3's interaction with the ribonucleotide reductase complex (RNR) and histone acetyltransferase KAT5

  • Characterization of how NME3 contributes to repair of both single and double-stranded DNA breaks

• Phosphohistidine signaling network:

  • Identification of proteins that are targets of NME3-mediated histidine phosphorylation

  • Development of tools to study protein histidine phosphorylation in live cells

• Mitochondrial membrane organization:

  • How NME3 hexamerization contributes to mitochondrial membrane tethering

  • Potential role in creating microdomains on the mitochondrial outer membrane

• Therapeutic targeting:

  • Development of small molecules that could modulate NME3 activity

  • Investigation of NME3 activation as a potential approach to enhance mitochondrial fusion in diseases with excessive mitochondrial fragmentation

How can researchers integrate multi-omics approaches to better understand NME3 function in complex biological systems?

Modern multi-omics approaches offer powerful tools to study NME3 in integrated biological systems:

• Integrative genomics approaches:

  • Combine whole genome/exome sequencing data from ciliopathy patients with functional studies of identified NME3 variants

  • Use GWAS data to identify potential associations between NME3 variants and disease phenotypes

• Transcriptomics integration:

  • RNA-seq analysis of NME3 knockout/knockdown models to identify affected pathways

  • Single-cell transcriptomics to identify cell type-specific effects of NME3 deficiency

  • Analysis of transcriptional changes in response to NME3 manipulation under different stress conditions

• Proteomics strategies:

  • Proximity labeling (BioID, APEX) with NME3 as bait to identify context-specific interaction partners

  • Quantitative phosphoproteomics to identify proteins with altered phosphorylation in NME3-deficient systems

  • Protein turnover studies to examine how NME3 affects stability of interacting proteins

• Metabolomics applications:

  • Targeted metabolomics to measure nucleotide levels and ratios in NME3-manipulated systems

  • Analysis of metabolic adaptations in response to NME3 deficiency under stress conditions

• Structural biology integration:

  • Cryo-EM studies of NME3 in complex with mitochondrial fusion machinery

  • Molecular dynamics simulations to understand how mutations affect NME3 function

• Systems biology modeling:

  • Mathematical modeling of how NME3 contributes to nucleotide homeostasis

  • Network analysis of NME3 interaction partners across different cellular compartments

By combining these approaches, researchers can develop a more comprehensive understanding of NME3's diverse functions and their relevance to human disease.