Recombinant Bovine NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 (NDUFA13)

What is NDUFA13 and what is its role in mitochondrial complex I?

NDUFA13 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13) is a supernumerary subunit of mitochondrial complex I with unique structural characteristics. It contains a transmembrane helix (TMH) structure that penetrates both Iα and Iλ structures within complex I, making it, to the best of current knowledge, the only protein with this feature . Originally identified as GRIM-19 (Gene associated with Retinoid-IFN-induced Mortality-19), NDUFA13 plays dual roles in energy metabolism and apoptosis regulation. Within the mitochondrial respiratory chain, it serves as a guardian that gauges electron flow across the electron transfer chain, ensuring energy transduction along the mitochondria in a safe and efficient manner . The unique position and structure of NDUFA13 suggest it forms a channel within complex I that interconnects the matrix with membrane interstitium.

How does the molecular structure of NDUFA13 relate to its function?

NDUFA13 possesses a distinct molecular architecture that directly influences its function within complex I. Analysis using MOE software and available structural data (PDB ID code: 5LDX) reveals that the first 33 amino acids of NDUFA13 extend along the dorsal side of the CoQ binding chamber after penetrating the inner membrane . These amino acids are positioned parallel to the last three FeS clusters (N2, N6b, and N6a), which are approximately 31 Å apart . The enlarged tail of NDUFA13 remains on the intermembrane side of ND1 and ND2 .

This unique positioning is critical for maintaining electron flow integrity. When NDUFA13 is moderately down-regulated, it creates a structural substrate allowing for an electron leak, resulting in continuous generation of small amounts of H₂O₂ . The importance of specific structural domains has been demonstrated through experiments with truncated NDUFA13 mutants, where deletion of amino acids 40-50 prevented proper mitochondrial localization and function, while deletions in other regions had minimal impact .

What are the key challenges in studying NDUFA13 function?

Investigating NDUFA13 presents several methodological challenges:

• Its integration within the large multi-subunit complex I makes isolation of native NDUFA13 difficult

• The protein's transmembrane domain creates challenges for recombinant expression and purification

• Distinguishing between structural and functional roles requires sophisticated experimental approaches

• Measuring the subtle effects on ROS generation necessitates sensitive and specific detection methods

• Achieving appropriate levels of NDUFA13 modulation is critical, as moderate down-regulation (30%) produces protective effects, while severe down-regulation (60%) is detrimental

These challenges have been addressed through the development of conditional knockout models, site-directed mutagenesis approaches, and advanced techniques for measuring ROS species .

How can researchers generate NDUFA13 knockout or knockdown models?

Several complementary approaches for NDUFA13 modulation have been documented:

• Cardiac-specific conditional knockout mice:
  Liu et al. generated cardiac-specific tamoxifen-inducible NDUFA13 knockout mice using Cre-loxP technology, allowing for tissue-specific and temporally controlled deletion of NDUFA13 .

• siRNA knockdown in cell culture:
  H9C2 cells transfected with NDUFA13-targeting siRNA at concentrations of 100 μmol/L and 200 μmol/L resulted in approximately 30% and 60% decreases in NDUFA13 expression, respectively .

• Adenoviral-mediated knockdown in primary cardiomyocytes:
  Neonatal mouse cardiomyocytes (NMCMs) isolated from NDUFA13 flox/flox mice were transfected with adenovirus containing Myh6-Cre to deplete endogenous NDUFA13 .

• Expression of truncated NDUFA13 mutants:
  Various adenoviruses containing different truncated NDUFA13 mutants were designed to investigate structure-function relationships:

  • Ad-1 (deletion of amino acids 40-50)

  • Ad-2 (deletion of amino acids 70-80)

  • Ad-3 (deletion of amino acids 110-120)

  • Ad-NDUFA13 (wild-type full-length NDUFA13)

  • Ad-Vector (empty vector control)

These models provide valuable tools for investigating the functional consequences of NDUFA13 down-regulation or structural modification in different experimental contexts.

What techniques are most effective for measuring ROS generation in NDUFA13 studies?

Multiple complementary techniques have proven effective for comprehensive ROS profiling:

• Site-specific H₂O₂ detection:

  • Cyto-HyPer and mito-HyPer probes enable detection of H₂O₂ in cytosol and mitochondria, respectively

  • These genetically encoded fluorescent sensors allow real-time monitoring of H₂O₂ levels in specific subcellular compartments

• Direct measurement of H₂O₂ from isolated mitochondria:

  • Amplex Red assay reacts with H₂O₂ in the presence of peroxidase to produce highly fluorescent resorufin

  • This method provided direct evidence of H₂O₂ generation following NDUFA13 knockdown

• Superoxide detection within mitochondria:

  • MitoSOX Red, a mitochondria-targeted dihydroethidium-based probe that becomes fluorescent upon oxidation by superoxide

  • This technique confirmed that moderate NDUFA13 knockdown did not increase superoxide generation at basal state

• Flow cytometry analysis:

  • Used in conjunction with specific fluorescent probes to quantify ROS levels across cell populations

  • Allows statistical analysis of ROS distribution within heterogeneous samples

The combination of these techniques is essential for distinguishing between different ROS species, which may have distinct biological effects and signaling roles in the context of NDUFA13 modulation.

How can researchers assess mitochondrial function in NDUFA13-modified systems?

Comprehensive assessment of mitochondrial function requires multiple methodological approaches:

• High-resolution respirometry:

  • The Oxygraph-2k (O2k; OROBOROS Instruments) enables precise measurement of mitochondrial respiration

  • Sequential addition of substrates and inhibitors allows determination of complex I, II, and IV respiration

  • This approach provides detailed analysis of electron transport chain function

• Mitochondrial membrane potential (MMP) measurement:

  • TMRM (tetramethylrhodamine methyl ester) staining followed by fluorescence intensity measurement or flow cytometry

  • This technique revealed that moderate NDUFA13 knockdown (30%) did not affect MMP, while severe knockdown (60%) impaired MMP

• Oxygen consumption rate (OCR) measurement:

  • NDUFA13 heterozygous knockout mice showed preserved capacity of oxygen consumption rate driven by complex I and II substrates

  • This matched the oxygen consumption driven by electron donors of TMPD+ascorbate

• Cytochrome C release assay:

  • Western blot analysis of cytosolic fractions detects cytochrome C release from mitochondria

  • This serves as an indicator of mitochondrial outer membrane permeabilization during apoptosis

These complementary approaches enable researchers to distinguish between effects on electron transport, membrane integrity, and apoptotic signaling in NDUFA13-modified systems.

How does NDUFA13 expression level influence ROS generation patterns?

NDUFA13 expression levels have distinct effects on ROS generation patterns as demonstrated in multiple experimental models:

• Moderate down-regulation of NDUFA13 (approximately 30% reduction):

  • Creates an electron leak within complex I

  • Results in a mild increase in cytoplasm-localized H₂O₂, but not superoxide

  • Does not affect mitochondrial membrane potential (MMP)

  • H₂O₂ serves as a second messenger activating protective signaling pathways

• Severe down-regulation of NDUFA13 (approximately 60% reduction):

  • Impairs mitochondrial membrane potential

  • Fails to elicit protection against hypoxia/reoxygenation-induced apoptosis

  • Does not produce the beneficial H₂O₂ signaling seen with moderate knockdown

• Normal NDUFA13 expression:

  • Maintains electron flow integrity through complex I

  • Prevents electron leak and subsequent H₂O₂ generation

  • Does not activate protective signaling pathways involving STAT3 and Bcl-2

These findings suggest that NDUFA13 functions as a "guardian" that gauges electron flow across the electron transfer chain, and its partial loss creates a specific ROS profile that can be either protective or harmful depending on the degree of downregulation.

What is the relationship between NDUFA13, H₂O₂ generation, and STAT3 signaling?

Research has elucidated a sophisticated signaling pathway linking NDUFA13, H₂O₂ generation, and STAT3 activation:

• Moderate down-regulation of NDUFA13 creates an electron leak within complex I, resulting in mild increase in cytosolic H₂O₂

• This increased H₂O₂ serves as a second messenger leading to:

  • Increased expression of peroxiredoxin 2 (PRX2)

  • PRX2-mediated STAT3 dimerization (phosphorylation at Tyr705)

  • Activation of antiapoptotic signaling

• Activated STAT3 then enhances Bcl-2 expression, which:

  • Inhibits mitochondrial outer membrane permeabilization

  • Prevents cytochrome C release

  • Suppresses activation of caspase-9 and caspase-3

• This signaling cascade ultimately results in decreased apoptosis and protection against ischemia-reperfusion injury

This protective effect was abolished by either knocking down STAT3 or by inhibiting H₂O₂ generation, confirming the essential role of the H₂O₂-STAT3-Bcl-2 pathway in mediating the protective effects of moderate NDUFA13 down-regulation.

How do different ROS species generated in relation to NDUFA13 affect cellular outcomes?

Different ROS species exert distinct effects on cellular outcomes in the context of NDUFA13 function:

The interrelationship between these ROS species reveals a sophisticated regulatory mechanism:

• The mild increase in H₂O₂ resulting from moderate NDUFA13 down-regulation at basal state activates protective mechanisms that prevent the superoxide burst during subsequent ischemia-reperfusion

• This represents a form of mitochondrial preconditioning where a mild stress (H₂O₂) protects against a severe stress (superoxide burst)

• Under normal oxygen supply, the short electronic effect of FMN is surpassed by the electron-withdrawing ability of downstream FeS clusters, but during oxygen deprivation and subsequent reperfusion, this balance is disrupted, leading to superoxide generation

This differential effect of ROS species highlights the complexity of redox signaling in mitochondrial biology and the specific role of NDUFA13 in orchestrating ROS profiles.

How does NDUFA13 modulation protect against ischemia-reperfusion injury?

Moderate down-regulation of NDUFA13 protects against ischemia-reperfusion (I/R) injury through several interconnected mechanisms:

These findings were validated across multiple experimental models, demonstrating the robust nature of this protective mechanism.

What cardiac phenotypes are associated with NDUFA13 modulation?

The effects of NDUFA13 modulation on cardiac phenotypes depend on the extent of down-regulation and the presence of stress conditions:

• Basal cardiac phenotype in moderate NDUFA13 down-regulation:

  • Cardiac-specific heterozygous knockout (cHet) mice exhibited normal cardiac morphology and function in the basal state

  • Echocardiographic parameters including interventricular septum dimensions (IVS), left ventricular internal dimensions (LVID), left ventricular posterior wall (LVPW), ejection fraction (EF), and fractional shortening (FS) showed no significant differences between cHet and control mice

• Response to ischemia-reperfusion injury:

  • cHet mice showed significantly decreased infarct size compared to control mice

  • Reduced apoptosis at the peri-infarct area

  • Decreased cleaved caspase-3 expression and cytochrome C release

The cardiac phenotypes associated with NDUFA13 modulation can be summarized in the following table:

These findings demonstrate that moderate NDUFA13 down-regulation provides significant cardioprotection without compromising baseline cardiac function.

What is the connection between NDUFA13, apoptosis, and cancer biology?

NDUFA13, originally identified as GRIM-19 (Gene associated with Retinoid-IFN-induced Mortality-19), has important connections to both apoptosis and cancer biology:

This connection between mitochondrial function, ROS signaling, and cell death regulation places NDUFA13 at the intersection of energy metabolism and cell survival decisions, with important implications for both cardiac protection and cancer biology.

How can recombinant NDUFA13 be used for structural and functional studies?

Recombinant NDUFA13 provides valuable tools for advanced structural and functional studies:

• Structure-function relationship studies:

  • Recombinant full-length bovine NDUFA13 protein (amino acids 2-144) with His-tag facilitates crystallography and structural analysis

  • Comparison with truncated NDUFA13 mutants allows investigation of specific domain functions

  • Site-directed mutagenesis of key residues can identify critical amino acids for NDUFA13 function

• Protein-protein interaction studies:

  • His-tagged recombinant NDUFA13 enables pull-down assays to identify interaction partners

  • This approach helps elucidate NDUFA13's role in complex I assembly and its interactions with other mitochondrial proteins

  • Cross-linking studies with purified recombinant protein can reveal spatial relationships within complex I

• In vitro reconstitution experiments:

  • Recombinant NDUFA13 can reconstitute complex I activity in NDUFA13-depleted mitochondria

  • This allows assessment of the protein's role in electron transfer and ROS generation

  • Different concentrations of recombinant protein can mimic the effects of varying NDUFA13 expression levels

• Development of specific antibodies and probes:

  • Purified recombinant NDUFA13 can generate specific antibodies for immunolocalization studies

  • Fluorescently labeled recombinant protein can serve as a probe for studying NDUFA13 trafficking and localization in live cells

These applications enable detailed investigation of NDUFA13's structure, interactions, and functions in various experimental settings.

What are the limitations of current NDUFA13 research methodologies?

Despite significant advances, NDUFA13 research faces several methodological limitations:

Addressing these limitations will require development of new techniques for fine-tuned protein modulation, improved real-time monitoring of ROS dynamics, and integration of structural and functional data across scales.

What future research directions might advance our understanding of NDUFA13 function?

Several promising research directions could significantly advance understanding of NDUFA13 function:

• Advanced structural studies:

  • Cryo-electron microscopy of complex I with various levels of NDUFA13 expression

  • Molecular dynamics simulations to model electron transfer pathways

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

• Novel genetic approaches:

  • CRISPR-based techniques for precise modulation of NDUFA13 expression

  • Development of knock-in models with structure-specific mutations

  • Tissue-specific inducible expression systems for temporal control

• Real-time monitoring technologies:

  • Development of NDUFA13-specific biosensors for real-time tracking of protein interactions

  • Integration of ROS-sensitive probes with mitochondrial function measurements

  • Live-cell imaging combined with high-resolution respirometry

• Systems biology approaches:

  • Multi-omics analysis of NDUFA13-modulated systems

  • Network modeling of NDUFA13-dependent signaling pathways

  • Machine learning applications for predicting context-dependent NDUFA13 effects

• Translational research directions:

  • Testing NDUFA13 modulation in large animal models of myocardial infarction

  • Development of pharmacological modulators of NDUFA13 function

  • Investigation of NDUFA13 polymorphisms associated with cardiovascular disease susceptibility

• Exploration of NDUFA13 in other tissues and disease contexts:

  • Examining the role of NDUFA13 in neurodegeneration, diabetes, and aging

  • Investigating tissue-specific differences in NDUFA13 function

  • Understanding the dual role of NDUFA13 in cancer and cardioprotection

These research directions would collectively enhance our understanding of this multifunctional protein and potentially lead to novel therapeutic strategies for cardiovascular protection.