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

What is NDUFA13 and what are its primary functions in cellular metabolism?

NDUFA13 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) that plays a crucial role in electron transfer from NADH to the respiratory chain. The protein is required for complex I assembly and electron transfer activity. It functions as an electron acceptor, with ubiquinone believed to be the immediate electron acceptor for the enzyme. NDUFA13 also has significant non-metabolic functions, including binding to signal transducers and activators of transcription 3 (STAT3) transcription factor, and it can function as a tumor suppressor . Additionally, it is involved in interferon/all-trans-retinoic acid (IFN/RA) induced cell death .

How does the molecular structure of NDUFA13 relate to its function in mitochondrial complex I?

NDUFA13 possesses a unique molecular structure with a physical location very close to the FeS clusters with low electrochemical potentials within complex I . This structural positioning is critical to its function, as it helps maintain the integrity of electron flow through the respiratory chain. The protein's structure enables it to participate in complex I assembly and stability while also supporting its secondary roles in cell signaling and apoptotic regulation. The proximity to FeS clusters with low electrochemical potentials makes NDUFA13 particularly interesting for studying electron leak phenomena and subsequent reactive oxygen species (ROS) generation within the mitochondrial respiratory chain .

What are the recommended storage and reconstitution protocols for recombinant NDUFA13 proteins?

For optimal stability, recombinant NDUFA13 proteins should be stored at -20°C/-80°C. The shelf life of liquid form is typically 6 months, while lyophilized forms can maintain stability for up to 12 months at these temperatures . For reconstitution, it is recommended to briefly centrifuge the vial before opening to bring contents to the bottom and then reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Addition of 5-50% glycerol (final concentration) is advised for long-term storage, with 50% being the standard recommendation . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided to maintain protein integrity .

How can NDUFA13 knockout models be effectively generated for studying mitochondrial dysfunction?

Creating effective NDUFA13 knockout models requires careful consideration of the complete versus partial knockout approach, as complete knockout may be embryonically lethal due to NDUFA13's essential functions. Cardiac-specific tamoxifen-inducible NDUFA13 knockout mice have been successfully generated to study the protein's role in cardiac function and ischemia-reperfusion injury responses . The research demonstrated that cardiac-specific heterozygous knockout (cHet) mice exhibited normal cardiac morphology and function in the basal state but were more resistant to apoptosis when exposed to ischemia-reperfusion injury .

For effective knockout model generation, researchers should:

• Consider conditional knockout approaches using Cre-loxP systems to avoid embryonic lethality

• Design tissue-specific promoters to target the knockout to relevant tissues

• Use inducible systems (e.g., tamoxifen-inducible) to control the timing of gene deletion

• Validate knockout efficiency at both mRNA and protein levels

• Characterize the functional consequences through measurements of complex I activity, oxygen consumption rates, and ROS production

What methodologies are most effective for investigating the role of NDUFA13 in reactive oxygen species (ROS) generation?

The investigation of NDUFA13's role in ROS generation requires a multi-faceted approach that combines genetic manipulation with sophisticated biochemical and imaging techniques. Based on research findings, NDUFA13 down-regulation creates a leak within complex I that can result in increased H₂O₂ production without affecting superoxide levels .

Recommended methodologies include:

• Genetic Manipulation Approaches:

  • Generate heterozygous knockout models rather than complete knockouts to study the physiological relevance of moderate NDUFA13 down-regulation

  • Use CRISPR-Cas9 or RNA interference techniques for targeted gene modification or silencing

• ROS Detection Methods:

  • Employ fluorescent probes specific for different ROS species (H₂O₂ vs. superoxide) with subcellular localization capabilities

  • Utilize genetically encoded ROS sensors targeted to different cellular compartments

  • Use high-resolution confocal microscopy to distinguish between mitochondrial and cytoplasmic ROS

• Functional Assessments:

  • Measure oxygen consumption rates using high-resolution respirometry or Seahorse technology

  • Assess complex I activity through spectrophotometric assays

  • Evaluate mitochondrial membrane potential using potential-sensitive dyes

• Molecular Signaling Analysis:

  • Investigate STAT3 dimerization and phosphorylation as downstream effects of NDUFA13-mediated ROS production

  • Assess antiapoptotic gene expression profiles associated with altered NDUFA13 levels

How does NDUFA13 promoter methylation influence gene expression and what are the implications for cancer research?

NDUFA13 promoter hypermethylation represents a significant epigenetic mechanism driving downregulation of this gene in multiple cancers . This methylation-driven silencing has particularly strong implications for breast cancer development, where decreased NDUFA13 expression leads to increased cell proliferation .

For investigating NDUFA13 methylation in cancer research, the following methodological approaches are recommended:

• Methylation Analysis Techniques:

  • Bisulfite sequencing to identify specific CpG sites with differential methylation

  • Methylation-specific PCR for targeted analysis of the NDUFA13 promoter region

  • Next-generation sequencing approaches for genome-wide methylation profiling

• Functional Validation:

  • Methylation inhibitor treatments (e.g., 5-azacytidine) to confirm reversibility of silencing

  • Luciferase reporter assays with methylated versus unmethylated promoter constructs

  • CRISPR-dCas9 epigenetic editing to manipulate methylation at specific sites

• Clinical Correlation Studies:

  • Analysis of NDUFA13 methylation patterns across different cancer types and stages

  • Correlation of methylation status with patient prognosis and treatment response

  • Development of methylation-based biomarkers for early cancer detection

The hypermethylated region identified within the NDUFA13 promoter is located approximately 130 bp from the transcription start site, which is particularly relevant for transcriptional control .

What is the relationship between NDUFA13 mutations and Leigh syndrome, and how can this be studied experimentally?

NDUFA13 mutations have been identified as causative factors in Leigh syndrome (LS), an early-onset mitochondrial encephalopathy characterized by bilateral symmetric lesions in the basal ganglia and cerebral stem . Novel biallelic variants in the NDUFA13 gene have been associated with isolated complex I deficiency in skeletal muscle .

For experimental investigation of NDUFA13-associated Leigh syndrome:

• Genetic Screening Approaches:

  • Next-generation sequencing panels targeting known mitochondrial disease genes

  • Whole-exome sequencing for novel variant discovery

  • Functional prediction of variant pathogenicity using in silico tools

• Functional Characterization:

  • Assessment of OXPHOS function in patient-derived fibroblasts

  • Measurement of delayed cell growth, enzyme activities, oxygen consumption, and ATP production

  • Quantification of NDUFA13 protein levels, complex I assembly, and respirasome formation

• Disease Modeling:

  • Generation of patient-specific induced pluripotent stem cells (iPSCs)

  • Differentiation of iPSCs into relevant cell types (neurons, cardiomyocytes)

  • CRISPR-based introduction of specific mutations into cellular or animal models

The clinical presentation of NDUFA13-associated Leigh syndrome can vary significantly between families, with some patients presenting with predominantly neurosensorial symptoms and others showing LS lesions in brain MRI, mild hypertrophic cardiomyopathy, and progressive spastic tetraparesis .

How do NDUFA13 variants affect oxidative phosphorylation and what are the downstream consequences?

NDUFA13 variants can significantly impair oxidative phosphorylation (OXPHOS) with specific effects on complex I function and broader mitochondrial energy metabolism. Patient-derived cells carrying NDUFA13 mutations demonstrate several key OXPHOS abnormalities:

• Primary OXPHOS Defects:

  • Isolated complex I enzyme deficiency

  • Decreased basal and maximal oxygen consumption

  • Reduced ATP production capacity

  • Diminished levels of assembled complex I and respirasomes

• Cellular Consequences:

  • Delayed cell growth and proliferation

  • Altered mitochondrial morphology and distribution

  • Compensatory metabolic adaptations (glycolysis upregulation)

  • ROS production changes affecting cellular signaling

These OXPHOS defects ultimately contribute to tissue-specific manifestations of disease, particularly affecting high-energy tissues such as the brain, heart, and skeletal muscle .

Methodological approaches for investigating OXPHOS dysfunction in NDUFA13 variants include:

• Blue Native PAGE for assessment of respiratory chain complex assembly

• High-resolution respirometry for detailed oxygen consumption analysis

• ATP production assays under various substrate conditions

• Mitochondrial network morphology assessment using confocal microscopy

What strategies can be employed to modulate NDUFA13 activity for potential therapeutic applications in cancer?

Given NDUFA13's dual role as both a mitochondrial complex I component and a tumor suppressor, several therapeutic strategies can be explored:

• Epigenetic Modification Approaches:

  • DNA methyltransferase inhibitors to reverse hypermethylation of the NDUFA13 promoter

  • Histone deacetylase inhibitors to promote open chromatin conformation and gene expression

  • Targeted epigenetic editing using CRISPR-dCas9 systems fused to demethylases

• Gene Therapy Approaches:

  • Viral vector-mediated NDUFA13 gene delivery to restore expression in cancer cells

  • mRNA-based therapies for transient expression enhancement

  • Small activating RNAs (saRNAs) targeting the NDUFA13 promoter

• Small Molecule Development:

  • Compounds that stabilize the NDUFA13 protein or enhance its integration into complex I

  • Molecules that mimic NDUFA13's tumor suppressor functions

  • Drugs that selectively target cancer cells with NDUFA13 deficiency

• Immunotherapy Approaches:

  • Development of cancer vaccines targeting cells with altered NDUFA13 expression

  • Chimeric antigen receptor T-cell therapy directed against surface markers upregulated in NDUFA13-deficient cells

Research into these therapeutic approaches should consider the tissue-specific effects of NDUFA13 modulation and potential off-target effects on mitochondrial function in healthy tissues.

How can NDUFA13-mediated ROS signaling be targeted to protect against ischemia-reperfusion injury?

Research has demonstrated that moderate down-regulation of NDUFA13 creates a mild increase in H₂O₂ production that activates protective STAT3 signaling and reduces vulnerability to ischemia-reperfusion injury . This presents an intriguing therapeutic target for cardioprotection and potentially other ischemic conditions.

Methodological approaches for harnessing NDUFA13-mediated ROS signaling include:

What are the key considerations for producing and purifying recombinant NDUFA13 for structural and functional studies?

Producing high-quality recombinant NDUFA13 requires careful attention to several technical aspects:

• Expression System Selection:

  • E. coli systems are commonly used for basic recombinant NDUFA13 production

  • Mammalian expression systems may be preferred for studies requiring post-translational modifications

  • Insect cell systems offer a compromise between bacterial and mammalian systems

• Purification Strategy Optimization:

  • Addition of appropriate tags (His, GST, etc.) to facilitate purification while minimizing interference with function

  • Implementation of multiple purification steps to achieve >85% purity as verified by SDS-PAGE

  • Tag removal considerations for functional studies

• Protein Stability Considerations:

  • Buffer optimization to maintain protein stability during purification and storage

  • Addition of glycerol (5-50%) for long-term storage

  • Aliquoting to avoid repeated freeze-thaw cycles

• Quality Control Measures:

  • Verification of protein identity through mass spectrometry

  • Assessment of folding status through circular dichroism

  • Functional validation through activity assays

• Special Considerations for Structural Studies:

  • Optimization of protein concentration and buffer conditions for crystallization

  • Consideration of co-crystallization with binding partners

  • NMR-based approaches for dynamic structural information

How should researchers approach experimental design when studying NDUFA13 interactions with STAT3 and other signaling proteins?

Investigation of NDUFA13's interactions with signaling proteins requires specialized experimental approaches:

• Protein-Protein Interaction Detection Methods:

  • Co-immunoprecipitation assays to verify interactions in cellular contexts

  • Yeast two-hybrid screening for identifying novel interaction partners

  • Proximity ligation assays for visualizing interactions in situ

  • FRET/BRET approaches for real-time interaction dynamics

• Structural Interaction Studies:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cryo-EM of protein complexes to determine three-dimensional arrangements

  • Molecular dynamics simulations to predict interaction mechanisms

• Functional Validation Approaches:

  • Mutagenesis of predicted interaction domains to disrupt specific binding

  • Peptide inhibitor design to competitively block interactions

  • Cellular assays measuring downstream signaling effects

• Temporal and Spatial Considerations:

  • Investigation of interaction dynamics during cellular stress responses

  • Subcellular localization studies to determine where interactions occur

  • Cell-cycle dependent interaction analysis

• Controls and Validation:

  • Use of NDUFA13 knockout cells as negative controls

  • Rescue experiments with wild-type versus mutant NDUFA13

  • Comparison with known interaction partners as positive controls