NME4 Human, Active

What is NME4 and what are its primary functions in human cells?

NME4, also known as NDPK-M or NM23-H4, is a nucleoside diphosphate kinase belonging to the nonmetastatic 23 (NM23) family. It specifically localizes to the mitochondrial intermembrane space via an N-terminal targeting sequence that requires cleavage for its catalytic activity. The protein binds to the inner mitochondrial membrane through interaction with anionic phospholipids, particularly cardiolipin .

The key functions of NME4 in mitochondrial physiology include:

• Transfer of oxidatively generated nucleoside triphosphates (NTPs) to different nucleoside diphosphates, primarily phosphorylating GDP to generate GTP for mitochondrial GTPases and other local energy requirements

• Promotion of intermembrane transfer of cardiolipin in mitochondria, which serves as either a pro-mitochondrial or pro-apoptotic signal depending on cellular context

What is the tissue distribution pattern of NME4 in humans?

Human NME4 shows a distinct expression pattern across different tissues. Based on current research findings, NME4 is:

• Highly expressed in liver tissue

• Expressed at intermediate levels in the heart and colon

• Expressed at low levels in the brain, testis, and peripheral leukocytes

This differential expression pattern suggests tissue-specific functions, with particularly important roles in metabolically active tissues like the liver, which aligns with findings regarding its involvement in lipid metabolism disorders.

How does NME4 differ from other members of the NM23 family?

While this specific comparison isn't directly addressed in the search results, we can infer that NME4's distinctive features include its mitochondrial localization and specific role in lipid metabolism. Unlike some other family members that may function in different cellular compartments, NME4's functions are specifically linked to mitochondrial energy metabolism and lipid processing, particularly in relation to coenzyme A metabolism and triglyceride regulation .

What are the most effective techniques for measuring NME4 activity in human samples?

Researchers investigating NME4 activity in human samples should consider a multi-method approach:

• Enzyme Activity Assays: Measure nucleoside diphosphate kinase activity by quantifying the phosphorylation of GDP to GTP in mitochondrial fractions.

• Protein Expression Analysis: Standard methods include:

  • Western blotting using validated anti-NME4 antibodies

  • qRT-PCR for mRNA expression levels

  • Immunohistochemistry for tissue localization studies

• Interaction Studies: To understand functional activity:

  • Tandem affinity purification mass spectrometry (TAP-MS) for stable protein interactions

  • Proximity-dependent biotinylating with mass spectrometry (TurboID-MS) to identify proteins in close proximity to NME4

When implementing these methods, researchers should consider mitochondrial isolation quality as critical for accurate results, given NME4's specific localization.

What genetic modification approaches are most suitable for NME4 functional studies?

Based on successful approaches documented in the research literature:

• CRISPR-Cas9 System: Effective for generating NME4 knockout cell lines, as demonstrated in hepatocyte research. This approach allows complete elimination of NME4 expression to study loss-of-function effects .

• RNA Interference: AAV-packaged shRNA targeting NME4 has been successfully used for in vivo liver-specific knockdown in mouse models. This approach resulted in significant reduction of NME4 expression in hepatocytes while minimizing effects on other tissues .

• Lentiviral Overexpression: Flag-tagged NME4 overexpression via lentiviral vectors has been utilized to study gain-of-function effects in primary hepatocytes .

For optimal results, researchers should consider cell type-specific effects and validation of knockdown/knockout efficiency through both protein and mRNA measurements.

What cellular models are most appropriate for studying human NME4 function?

Based on experimental evidence, researchers have successfully employed several models:

• Human Liver Cancer Cell Lines:

  • HepG2 and Bel-7402 cells (high endogenous NME4 expression)

  • SMMC-7721 and SK-Hep1 cells (low endogenous NME4 expression)

  These differential expression patterns allow researchers to select appropriate models depending on whether knockdown or overexpression approaches are preferred .

• Primary Mouse Hepatocytes: Isolated through two-step perfusion, these cells have been effectively used for NME4 overexpression studies and respond to palmitic acid/oleic acid treatment to model fatty acid loading .

• Mouse Hepa1-6 Cell Line: Another useful model for NME4 depletion studies in the context of lipid metabolism .

When selecting a model, researchers should consider the specific aspect of NME4 function under investigation, with hepatocyte models being particularly relevant for lipid metabolism studies.

How does NME4 contribute to nonalcoholic fatty liver disease (NAFLD) pathogenesis?

NME4 plays a significant role in NAFLD pathogenesis through several mechanisms:

• Upregulation in Disease State: NME4 expression is markedly upregulated in high-fat diet (HFD) mouse models in a time-dependent manner, with expression levels positively correlating with steatosis severity .

• Lipid Accumulation Mechanism: NME4 interacts with key enzymes in coenzyme A (CoA) metabolism, leading to increased levels of acetyl-CoA and malonyl-CoA. These metabolites are critical precursors for triglyceride synthesis, driving hepatic lipid accumulation .

• Cell-Specific Effects: Single-cell database analysis indicates NME4 is predominantly expressed in hepatocytes rather than other liver cell types, suggesting cell-type-specific mechanisms in NAFLD .

• Experimental Evidence: Knockdown of NME4 in mouse liver via AAV-shRNA significantly reduced:

  • Liver weight

  • Serum ALT levels (indicating reduced liver injury)

  • Hepatic steatosis (confirmed by H&E and Oil Red O staining)

These findings collectively demonstrate that NME4 promotes NAFLD progression primarily through its effects on hepatic triglyceride metabolism.

What lipidomic changes are associated with NME4 activation or inhibition?

NME4 manipulation significantly alters the hepatic lipidome as evidenced by targeted lipidomic analyses:

• NME4 Depletion Effects:

  • In mouse models, AAV-sh-NME4 infection resulted in 26 significantly downregulated lipids and 17 upregulated lipids

  • Triglycerides (TGs) constituted the majority of downregulated lipids

  • Biochemical assays confirmed significant decreases in TG levels

• NME4 Overexpression Effects:

  • Human liver cancer cell studies showed 21 significantly upregulated lipids with NME4 overexpression

  • Again, TGs were predominantly affected

  • Other affected lipid classes included diacylglycerols and cholesteryl esters

• Quantifiable Changes:

  • In vitro biochemical assays demonstrated decreased TG and cholesterol levels in NME4-knockout cells treated with palmitic/oleic acid

  • These effects were reversed with NME4 overexpression, confirming a direct relationship

These lipidomic alterations highlight NME4's specific role in regulating lipid metabolism, particularly triglyceride synthesis and accumulation.

What is the relationship between NME4 expression and metabolic stress responses?

The relationship between NME4 and metabolic stress appears complex:

• Stress-Induced Expression: NME4 expression increases in a concentration- and time-dependent manner when primary mouse hepatocytes are treated with palmitic acid/oleic acid mixtures, suggesting its upregulation is a response to lipid loading stress .

• Not Simply a Mitochondrial Stress Marker: While one might hypothesize that NME4 upregulation merely reflects mitochondrial stress, research findings challenge this interpretation. In HFD-fed mice with increased NME4 expression, genes associated with mitochondrial function (Ppargc1a, Ndufs7, Cox5a, and Cox8b) were actually slightly downregulated, indicating that NME4 upregulation is not simply a marker of enhanced mitochondrial function or general mitochondrial stress response .

• Specific Metabolic Pathway Involvement: Proteomic analysis of NME4-depleted cells revealed significant enrichment of proteins involved in lipid metabolism and lipid transport, suggesting NME4 has specific roles in these metabolic pathways rather than being a general stress response protein .

These findings suggest NME4 plays an active role in metabolic reprogramming during stress conditions rather than being merely a stress response marker.

How does the protein interaction network of NME4 influence its role in lipid metabolism?

The protein interaction network of NME4 provides crucial insights into its mechanistic role in lipid metabolism:

• Interaction Detection Methodology: Researchers have employed complementary approaches to identify NME4's protein interactions:

  • Tandem affinity purification mass spectrometry (TAP-MS) to identify stable protein interactions

  • Proximity-dependent biotinylating with mass spectrometry (TurboID-MS) to identify proteins in close proximity

• Mitochondrial Localization Importance: NME4's mitochondrial localization is critical for its interaction network. Wild-type NME4 primarily labels proteins localized to mitochondria, while a mutant lacking the mitochondrial localization sequence shows diminished ability to label proximal proteins .

• Key Interaction Partners: NME4 interacts with several key enzymes involved in coenzyme A (CoA) metabolism, which directly influences the levels of:

  • Acetyl-CoA

  • Malonyl-CoA

  These metabolites are major components of lipid synthesis pathways in the liver .

• Functional Consequences: These protein interactions ultimately result in increased triglyceride levels, promoting lipid accumulation in hepatocytes and driving NAFLD progression .

Understanding this interaction network provides potential targets for therapeutic intervention in metabolic disorders associated with dysregulated lipid metabolism.

What are the molecular mechanisms by which NME4 regulates mitochondrial function in different metabolic states?

NME4's regulation of mitochondrial function across metabolic states involves several mechanisms:

• Nucleotide Balance Regulation: As a nucleoside diphosphate kinase, NME4 facilitates the transfer of oxidatively generated NTPs to different nucleoside diphosphates, primarily converting GDP to GTP. This activity provides local fuel for mitochondrial GTPases, potentially influencing mitochondrial dynamics and function .

• Membrane Lipid Organization: NME4 promotes the intermembrane transfer of cardiolipin in mitochondria. Cardiolipin is a critical phospholipid for proper mitochondrial function, and its distribution can serve as either a pro-mitochondrial or pro-apoptotic signal depending on cellular context .

• Metabolic Intermediates Regulation: Through interactions with enzymes involved in CoA metabolism, NME4 influences levels of acetyl-CoA and malonyl-CoA. These intermediates not only serve as building blocks for lipids but also regulate various aspects of mitochondrial energy metabolism .

• Differential Responses to Nutritional Status: Under high-fat diet conditions, NME4 upregulation appears to drive metabolic reprogramming that favors lipid accumulation rather than oxidation, suggesting a role in adapting mitochondrial function to nutritional excess .

These mechanisms collectively highlight NME4's role as a metabolic switch that influences mitochondrial function in response to changing nutritional states.

How does post-translational modification affect NME4 activity and localization?

While the provided search results don't explicitly address post-translational modifications of NME4, we can infer several important aspects:

• N-terminal Processing: NME4 contains a specific N-terminal sequence that targets it to the mitochondrial intermembrane space. This sequence must be cleaved and removed to allow its catalytic activity, representing a critical post-translational modification essential for function .

• Membrane Association: NME4 binds to the inner mitochondrial membrane via interaction with anionic phospholipids, particularly cardiolipin. This association likely involves specific protein domains and potentially post-translational modifications that regulate membrane binding affinity .

• Activity Regulation: The catalytic activity of NME4 as a nucleoside diphosphate kinase may be subject to regulation through post-translational modifications, though specific details are not provided in the available search results.

Research on these modifications would be valuable for understanding how NME4 activity is regulated under different physiological and pathological conditions.

How should researchers address contradictory findings regarding NME4 function in different experimental models?

When confronted with contradictory findings regarding NME4 function across experimental models, researchers should systematically:

• Evaluate Model Appropriateness:

  • Consider tissue-specific expression patterns. NME4 is highly expressed in liver, moderately in heart and colon, and at low levels in brain, testis, and peripheral leukocytes .

  • Select models that reflect the tissue of interest. For liver-related functions, HepG2, Bel-7402, or primary hepatocytes may be more relevant than other cell types .

• Account for Experimental Conditions:

  • Nutritional state significantly impacts NME4 function. High-fat diet conditions upregulate NME4 in a time-dependent manner .

  • Consider whether palmitic/oleic acid treatment was used to induce lipid loading, as this affects NME4 expression and function .

• Assess Knockdown/Overexpression Efficiency:

  • Verify NME4 manipulation at both mRNA and protein levels

  • Consider the method used (CRISPR-Cas9, shRNA, lentiviral overexpression) and its efficiency in the specific model

• Evaluate Endpoint Measurements:

  • For lipid metabolism studies, confirm whether appropriate methods were used (Oil Red O staining, lipidomic analysis, biochemical TG measurements)

  • Consider whether single endpoints or comprehensive analyses were performed

• Analyze Experimental Controls:

  • For in vivo studies, verify whether appropriate controls for viral delivery were included

  • In overexpression studies, confirm whether empty vector controls were used

By systematically addressing these factors, researchers can better reconcile contradictory findings and determine the context-specific functions of NME4.

What methodological considerations are critical when analyzing NME4's role in lipid metabolism?

When analyzing NME4's role in lipid metabolism, several critical methodological considerations should be addressed:

• Subcellular Fractionation Quality:

  • Given NME4's specific localization to the mitochondrial intermembrane space, high-quality mitochondrial isolation is essential

  • Contamination from other cellular compartments can significantly confound results

• Model Selection and Relevance:

  • Prioritize models with physiologically relevant NME4 expression levels

  • Consider using both cell lines with naturally high NME4 expression (HepG2, Bel-7402) and those with low expression (SMMC-7721, SK-Hep1) for complementary approaches

• Lipid Analysis Comprehensiveness:

  • Combine multiple methodologies:

    • Histological techniques (H&E and Oil Red O staining)

    • Biochemical assays for TG and cholesterol quantification

    • Comprehensive lipidomic profiling to identify specific lipid species affected

• Temporal Considerations:

  • NME4 expression changes in a time-dependent manner in response to HFD

  • Design experiments with appropriate time points to capture dynamic changes

• Intervention Specificity:

  • When using viral vectors for in vivo studies, verify tissue-specific targeting

  • Confirm effects on target tissue (liver) versus non-target tissues (e.g., adipose)

• Metabolic Context:

  • Account for nutritional state and feeding conditions

  • Consider fasting/fed state when collecting samples

Addressing these methodological considerations will enhance the reliability and reproducibility of findings regarding NME4's role in lipid metabolism.

How can researchers distinguish direct effects of NME4 from secondary metabolic adaptations?

Distinguishing direct NME4 effects from secondary metabolic adaptations requires careful experimental design:

• Time-Course Experiments:

  • Implement early time points after NME4 manipulation to capture immediate effects before secondary adaptations occur

  • Compare with later time points to identify progressive changes that may represent adaptive responses

• Protein Interaction Studies:

  • Utilize techniques like TAP-MS and TurboID-MS to identify direct protein interactions

  • Focus on consistent interaction partners across different experimental conditions to identify core NME4 functions

• Metabolite Flux Analysis:

  • Employ isotope-labeled metabolic precursors to track the immediate impact of NME4 on specific metabolic pathways

  • Analyze incorporation rates into downstream metabolites to determine direct metabolic effects

• Rescue Experiments:

  • After NME4 knockdown, introduce wild-type or mutant NME4 variants to determine which protein domains or functions restore the original phenotype

  • Use point mutations that affect specific functions while preserving others to dissect multifunctional aspects

• Acute vs. Chronic Manipulation:

  • Compare acute (e.g., inducible systems) versus chronic NME4 manipulation

  • Acute effects are more likely to represent direct consequences, while chronic effects may include compensatory mechanisms

• Cross-Reference with Multi-Omics Data:

  • Integrate proteomic, lipidomic, and transcriptomic data to distinguish primary from secondary effects

  • Primary effects should be consistent across multiple types of analyses and experimental models

This multi-layered approach allows researchers to confidently attribute observed phenotypes to direct NME4 activity versus compensatory responses.

What are promising therapeutic strategies targeting NME4 for metabolic disorders?

Based on current understanding of NME4 biology, several promising therapeutic strategies emerge:

• NME4 Inhibition Approaches:

  • RNA interference: AAV-delivered shRNA targeting hepatic NME4 has shown efficacy in reducing liver steatosis in mouse models, suggesting therapeutic potential

  • Small molecule inhibitors: Development of compounds targeting NME4's nucleoside diphosphate kinase activity or protein interactions could provide pharmacological options

• Targeting Protein-Protein Interactions:

  • Disrupting specific interactions between NME4 and key enzymes in CoA metabolism could selectively inhibit lipogenic effects while preserving other functions

  • Peptide-based inhibitors designed to interfere with these interactions represent a potential approach

• Metabolic Bypass Strategies:

  • Interventions targeting downstream metabolic pathways affected by NME4 overactivity, particularly those involving acetyl-CoA and malonyl-CoA metabolism

  • This could include enhancing fatty acid oxidation pathways to counter NME4-mediated lipid accumulation

• Tissue-Specific Delivery Approaches:

  • Given NME4's expression in multiple tissues, liver-targeted delivery systems (similar to the AAV approach used experimentally) could maximize therapeutic efficacy while minimizing off-target effects

  • Hepatocyte-specific promoters could drive expression of therapeutic constructs

• Combination Therapies:

  • Combining NME4 inhibition with other metabolic interventions could provide synergistic benefits

  • Potential partners include PPAR agonists or other established metabolic disease treatments

Each of these approaches warrants further investigation to determine safety, efficacy, and translational potential.

What aspects of NME4 regulation remain unexplored in current research?

Despite significant advances in understanding NME4 function, several important regulatory aspects remain underexplored:

• Transcriptional Regulation:

  • The transcription factors and regulatory elements controlling NME4 expression, particularly in response to metabolic stress

  • Epigenetic mechanisms that might influence tissue-specific expression patterns

• Post-Translational Modifications:

  • Comprehensive characterization of modifications beyond the known N-terminal processing

  • Potential phosphorylation, acetylation, or other modifications that might regulate activity or localization

  • How these modifications change under different metabolic conditions

• Mitochondrial Import and Processing:

  • Detailed mechanisms of NME4 import into mitochondria

  • Identity of proteases responsible for cleaving the N-terminal targeting sequence

  • Regulation of these processes under different cellular conditions

• Integration with Cellular Signaling Networks:

  • How NME4 activity responds to broader cellular signaling pathways

  • Potential crosstalk with insulin signaling, mTOR, AMPK, or other metabolic regulatory networks

• Circadian and Hormonal Regulation:

  • Whether NME4 expression or activity follows circadian patterns

  • Effects of key metabolic hormones on NME4 function

• Genetic Variants in Human Populations:

  • Prevalence and significance of NME4 polymorphisms

  • Association of these variants with metabolic disease susceptibility

Addressing these knowledge gaps would provide a more comprehensive understanding of NME4 biology and potential therapeutic approaches.

How might NME4 function interact with other mitochondrial proteins in metabolic disease contexts?

The interaction between NME4 and other mitochondrial proteins in metabolic disease likely involves complex networks:

Understanding these interaction networks could reveal new therapeutic targets and explain how NME4 dysregulation contributes to the complex pathophysiology of metabolic diseases.

Table 1: Expression of NME4 in Human Tissues

Table 2: Key Protein Interactions of NME4 Identified Through Proximity Labeling

Table 3: Effects of NME4 Manipulation on Lipid Profiles