Recombinant Mouse NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 (Ndufa13)

What is Ndufa13 and what is its cellular localization?

Ndufa13 (also known as GRIM-19) is an accessory subunit of NADH dehydrogenase (ubiquinone), which constitutes complex I of the mitochondrial electron transport chain. This protein is specifically located in the mitochondrial inner membrane, where it contributes to the largest of the five complexes in the respiratory chain. Ndufa13 is one of approximately 31 hydrophobic subunits that form the transmembrane region of Complex I . The protein exhibits a unique molecular structure with an N-terminal hydrophobic domain that forms an alpha helix spanning the inner mitochondrial membrane, while its C-terminal hydrophilic domain interacts with the globular subunits of Complex I . Unlike core subunits, Ndufa13 is considered an accessory subunit that is not directly involved in the catalytic activity of the complex but plays critical roles in complex stability and regulation.

What is the molecular structure of Ndufa13?

Ndufa13 possesses a distinctive two-domain structure that is highly conserved, suggesting its critical importance for protein function. The protein is approximately 17 kDa and composed of 144 amino acids in humans . Its structure is characterized by:

• An N-terminal hydrophobic domain that folds into an alpha helix, serving as an anchor within the inner mitochondrial membrane

• A C-terminal hydrophilic domain that interacts with the globular components of Complex I

• A predicted secondary structure primarily consisting of alpha helices, with the carboxy-terminal half having high potential to adopt a coiled-coil conformation

• An amino-terminal region containing a putative beta sheet rich in hydrophobic amino acids that may function as a mitochondrial import signal

Notably, Ndufa13 contains a transmembrane helix (TMH) structure that uniquely penetrates both Iα and Iλ, two important structural components within complex I . This distinctive structural feature positions Ndufa13 close to FeS clusters with low electrochemical potentials, making it potentially significant in electron transfer and ROS generation .

How does Ndufa13 contribute to mitochondrial function?

Ndufa13 contributes to mitochondrial function primarily through its role in complex I stability and potentially in regulating electron flow. While it is not directly involved in the catalytic transfer of electrons from NADH to ubiquinone, its strategic positioning near subunits with low electrochemical potentials makes it significant for maintaining proper electron flow .

When Ndufa13 expression is moderately down-regulated, it creates a specific type of electron leak within complex I that results in the generation of hydrogen peroxide (H₂O₂) but not superoxide . This controlled ROS production can actually serve beneficial signaling functions. For example, in cardiac tissue, the H₂O₂ generated acts as a second messenger responsible for STAT3 dimerization and activation of antiapoptotic signaling pathways . This ultimately protects against damage during ischemia-reperfusion events by suppressing superoxide burst and reducing infarct size .

What is known about NDUFA13 gene organization and expression?

In humans, the NDUFA13 gene is located on the short (p) arm of chromosome 19 at position 13.2 and spans approximately 11,995 base pairs . The gene encodes the 17 kDa Ndufa13 protein of 144 amino acids.

Expression of NDUFA13 has been found to be altered in various pathological conditions:

• Decreased expression has been reported in various tumors, with down-regulation rendering tumor cells more resistant to apoptosis and chemotherapy

• Monoallelic loss of NDUFA13 has been shown to promote tumorigenesis in mice, associated with decreased apoptosis

• Conversely, upregulation of NDUFA13 expression (such as through IFN/retinol administration in MCF-7 cells) has been shown to increase apoptosis by approximately 50%, indicating its proapoptotic effects

• Biallelic variants in NDUFA13 have been associated with a distinct clinical phenotype characterized by neurodevelopmental abnormalities, movement disorders, cerebellar ataxia, and epilepsy (OMIM #618249)

What mechanisms underlie electron leak from Ndufa13 within mitochondrial complex I?

The electron leak associated with Ndufa13 modification appears to be highly specific in both its mechanism and consequences. The unique position of Ndufa13 within complex I—specifically its proximity to FeS clusters with low electrochemical potentials—creates a specialized environment for controlled electron leakage when the protein is moderately down-regulated .

The mechanism involves:

This specific mechanism distinguishes Ndufa13-related electron leak from other forms of mitochondrial dysfunction. The electron leak created by moderate Ndufa13 down-regulation appears to be a controlled process that potentially serves physiological signaling functions rather than representing pathological dysfunction.

How does Ndufa13 deficiency affect apoptosis regulation?

The relationship between Ndufa13 expression and apoptosis regulation is complex and depends on the degree of deficiency:

The molecular pathway connecting Ndufa13 deficiency to apoptosis regulation involves ROS-mediated activation of STAT3. The H₂O₂ generated due to electron leak serves as a second messenger responsible for STAT3 dimerization and activation of antiapoptotic signaling .

What is the relationship between Ndufa13 and ROS generation?

Ndufa13 plays a unique role in reactive oxygen species (ROS) generation that differs significantly based on the degree of its down-regulation:

• Basal state with moderate down-regulation:

  • Creates a specific electron leak within complex I

  • Results in mild increase in cytoplasm-localized H₂O₂

  • Importantly, does not increase superoxide production

  • The H₂O₂ generated acts as a signaling molecule rather than a damaging agent

• During ischemia-reperfusion (I/R):

  • Moderate Ndufa13 down-regulation significantly suppresses the typical superoxide burst that occurs during I/R

  • This suppression is mediated by the activation of antiapoptotic signaling pathways initiated by the baseline H₂O₂ production

• With severe down-regulation:

  • May lead to more substantial mitochondrial dysfunction

  • Likely alters the ROS profile, potentially increasing harmful forms of ROS

  • Loses the protective signaling benefits observed with moderate down-regulation

Research using neonatal cardiomyocytes (NMCMs) with Ndufa13 deletion showed that at basal state, cells treated with Ad-Cre or Ad-NC (controls) had similar levels of superoxide, as detected with mitoSOX Red probes . This confirms that moderate Ndufa13 down-regulation specifically affects H₂O₂ production without significantly altering superoxide levels.

What clinical conditions are associated with NDUFA13 variants?

Biallelic variants in the NDUFA13 gene have been associated with a distinct clinical phenotype classified under OMIM #618249 . Based on current research, the clinical manifestations include:

• A spectrum of neurodevelopmental abnormalities

• Progressive complex movement disorders

• Cerebellar ataxia

• Neurosensory abnormalities

• Epilepsy

This condition belongs to the broader category of mitochondrial complex I deficiency disorders and appears to manifest as a form of Leigh syndrome. Researchers have identified at least 8 individuals from 7 independent families worldwide with biallelic variants in the NDUFA13 gene presenting with this clinical picture .

The clinical and molecular spectrum of NDUFA13-related complex I deficiency remains poorly characterized, as only two families have been reported in detail in the literature . Further research is needed to better understand the genotype-phenotype correlations, progression patterns, and potential therapeutic approaches for patients with these rare variants.

What approaches are optimal for generating Ndufa13 knockout models?

Based on successful experimental approaches documented in the literature, the following methods have proven effective for generating Ndufa13 knockout models:

• Cardiac-specific tamoxifen-inducible Ndufa13 knockout:

  • This approach allows for temporal control of Ndufa13 deletion

  • In published studies, researchers administered tamoxifen (40 mg/kg/d) for 5 consecutive days to achieve conditional knockout

  • This method enables the study of both heterozygous (cHet) and homozygous (cHomo) knockout effects

  • The time-dependent down-regulation can be assessed, with cHet mice showing moderate decrease by day 16 post-tamoxifen, while cHomo mice show moderate decrease as early as day 1 and approximately 80% decrease by day 16

• siRNA-mediated knockdown in cell culture:

  • Different doses of siRNA-NDUFA13 (100 μmol/L for moderate, 200 μmol/L for severe knockdown) have been used to achieve varying degrees of down-regulation

  • This approach allows for studying dose-dependent effects on mitochondrial function and cell survival

• Adenovirus-mediated Cre recombinase expression:

  • For studies using isolated neonatal cardiomyocytes (NMCMs) from floxed mice

  • Cells can be transfected with adenovirus containing Cre recombinase (Ad-Cre) or empty vector as control (Ad-NC)

  • This approach enables cell-specific deletion of Ndufa13 in primary culture systems

Each of these approaches has specific advantages depending on the research question. The tamoxifen-inducible system is particularly valuable for studying the physiological and pathological consequences of Ndufa13 deficiency in vivo, while the cell culture approaches offer more precise control over knockdown levels and are suitable for detailed mechanistic studies.

How can researchers accurately measure electron leak and ROS generation associated with Ndufa13 dysfunction?

Accurate measurement of electron leak and specific ROS species is crucial for understanding the functional consequences of Ndufa13 modification. The following methodological approaches have been validated:

When designing experiments to measure ROS production in Ndufa13-modified systems, it is important to:

• Include appropriate positive and negative controls

• Use multiple complementary methods to detect different ROS species

• Consider the subcellular localization of ROS (mitochondrial versus cytosolic)

• Account for potential compensatory mechanisms that may affect ROS levels

What techniques are effective for studying Ndufa13's role in cell signaling pathways?

To investigate how Ndufa13-mediated ROS production affects downstream signaling pathways, especially STAT3 activation, the following techniques have proven valuable:

• Western blotting for STAT3 dimerization and phosphorylation:

  • Allows quantification of total STAT3, phosphorylated STAT3, and STAT3 dimers

  • Non-reducing gel conditions can be used to preserve STAT3 dimers for analysis

  • Particularly important for confirming the link between H₂O₂ production and STAT3 activation

• Immunoprecipitation assays:

  • Can be used to detect protein-protein interactions involving Ndufa13

  • Useful for identifying binding partners that may mediate its effects on signaling pathways

• Transcriptional reporter assays:

  • Luciferase reporters containing STAT3-responsive elements

  • Allow functional assessment of STAT3 transcriptional activity downstream of Ndufa13 modulation

• RNA-seq or qPCR for target gene expression:

  • Enables identification of genes regulated by the Ndufa13-ROS-STAT3 axis

  • Provides insight into the broader cellular consequences of this signaling pathway

• Pharmacological interventions:

  • ROS scavengers can confirm the role of H₂O₂ in STAT3 activation

  • STAT3 inhibitors can validate the importance of this pathway in mediating the effects of Ndufa13 down-regulation

When designing experiments to study these signaling pathways, researchers should consider:

• Temporal dynamics of signaling activation

• Cell type-specific differences in signaling responses

• Potential cross-talk with other pathways

• The specificity of pharmacological inhibitors used

How should contradictory data regarding Ndufa13's role in apoptosis be reconciled?

Researchers may encounter seemingly contradictory findings regarding Ndufa13's role in apoptosis, with some studies suggesting pro-apoptotic functions and others indicating anti-apoptotic effects. These apparent contradictions can be reconciled by considering:

When evaluating apparently contradictory data, researchers should carefully consider these factors and design experiments that systematically vary the degree of Ndufa13 down-regulation, cell type, and experimental conditions to clarify the context-dependent nature of its effects on apoptosis.

What considerations are important when interpreting ROS measurements in Ndufa13-modified systems?

When interpreting ROS measurements in systems with modified Ndufa13 expression, researchers should consider:

• Specificity of ROS detection methods:

  • Different probes detect specific ROS species (superoxide, H₂O₂, hydroxyl radicals)

  • Ndufa13 deficiency appears to specifically increase H₂O₂ but not superoxide

  • Using multiple detection methods can provide more comprehensive understanding

• Subcellular localization of ROS:

  • Distinguish between mitochondrial and cytosolic ROS

  • Ndufa13 down-regulation leads to H₂O₂ that can exit mitochondria and act in the cytosol

  • Compartment-specific ROS may have different signaling consequences

• Temporal dynamics:

  • Baseline versus stress-induced ROS production may show different patterns

  • Acute versus chronic Ndufa13 deficiency may yield different ROS profiles

  • Consider measuring ROS at multiple time points after inducing Ndufa13 deficiency

• Magnitude of ROS increase:

  • Moderate increases may serve as signaling molecules

  • Large increases typically indicate oxidative stress and damage

  • Quantify changes relative to physiological baselines

• Downstream effects:

  • Assess oxidative damage markers (protein carbonylation, lipid peroxidation, DNA damage)

  • Evaluate activation of antioxidant defense mechanisms

  • Examine ROS-sensitive signaling pathways (e.g., STAT3, NF-κB)

A comprehensive approach to ROS assessment in Ndufa13-modified systems would include measurements of:

• Multiple ROS species (H₂O₂, superoxide, hydroxyl radicals)

• Compartment-specific ROS (mitochondrial versus cytosolic)

• Temporal dynamics of ROS production

• Oxidative damage markers

• Activation of ROS-sensitive signaling pathways

How can researchers distinguish between pathological and physiological consequences of Ndufa13 deficiency?

Distinguishing between pathological dysfunction and adaptive physiological responses in Ndufa13-deficient systems requires careful experimental design and interpretation:

• Assessment of mitochondrial function:

  • Measure key parameters such as:

    • ATP production

    • Oxygen consumption rate (OCR)

    • Mitochondrial membrane potential

    • Complex I activity

  • Preserved ATP levels and OCR with moderate Ndufa13 down-regulation suggest physiological adaptation rather than pathological dysfunction

• Evaluate cellular outcomes:

  • Cell viability and proliferation

  • Response to additional stressors (e.g., hypoxia, oxidative stress)

  • In cardiac models, moderate Ndufa13 down-regulation improved outcomes during I/R injury, indicating a beneficial adaptive response

• Time-course studies:

  • Short-term versus long-term consequences of Ndufa13 deficiency

  • Acute compensatory responses versus chronic pathological changes

  • Research has shown time-dependent effects, with cHomo mice exhibiting progressive decline in ATP levels while cHet mice maintained normal ATP content

• Dose-response relationships:

  • Systematically vary the degree of Ndufa13 down-regulation

  • Identify thresholds between adaptive and pathological responses

  • Data indicates that while moderate down-regulation (cHet) has beneficial effects, severe deficiency (cHomo) leads to progressive mitochondrial dysfunction

• Molecular signature analysis:

  • Transcriptomic or proteomic profiling to identify activation of:

    • Stress response pathways

    • Adaptive metabolic programs

    • Cell death pathways

  • Compare these signatures to known pathological states versus physiological adaptations

• In vivo phenotyping:

  • Assess organ function (e.g., cardiac function in heart-specific knockout models)

  • Examine for pathological changes at tissue and cellular levels

  • Evaluate response to physiological challenges

By integrating these approaches, researchers can develop a more nuanced understanding of when Ndufa13 deficiency represents a pathological state versus when it triggers beneficial adaptive responses.