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

What is NDUFA13 and what is its role in mitochondrial function?

NDUFA13 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13) is an accessory subunit of Complex I (NADH dehydrogenase) in the mitochondrial electron transport chain. In humans, it is encoded by the NDUFA13 gene located on chromosome 19p13.2, spanning approximately 11,995 base pairs . The gene produces a 17 kDa protein composed of 144 amino acids .

As a component of Complex I, NDUFA13 contributes to the largest of the five respiratory complexes in the mitochondrial inner membrane. While it is not directly involved in catalysis, it plays crucial structural and regulatory roles . Complex I as a whole facilitates the transfer of electrons from NADH to ubiquinone, which is essential for cellular energy production through oxidative phosphorylation .

NDUFA13 is also known by several alternative names including:

• Cell Death Regulatory Protein GRIM-19

• NADH-Ubiquinone Oxidoreductase B16.6 Subunit

• Complex I-B16.6

• CI-B16.6

• MC1DN28

What is the structural organization of NDUFA13?

NDUFA13 exhibits a distinctive two-domain structure that appears to be highly conserved:

• N-terminal hydrophobic domain:

  • Potential to fold into an alpha helix

  • Spans the inner mitochondrial membrane

  • Contains a putative beta sheet rich in hydrophobic amino acids

  • May serve as a mitochondrial import signal

• C-terminal hydrophilic domain:

  • Interacts with globular subunits of Complex I

  • Has high potential to adopt a coiled-coil form

How does NDUFA13 contribute to electron transport in mitochondria?

NDUFA13 is strategically positioned within Complex I near regions with lower electrochemical potentials, which may influence electron flow patterns . The human NDUFA13 contributes to Complex I function by:

• Facilitating electron transfer from NADH to ubiquinone

• Supporting the architectural integrity of Complex I

• Potentially regulating electron leak and subsequent reactive oxygen species (ROS) generation

In the electron transport chain, NADH binds to Complex I and transfers two electrons to the flavin mononucleotide (FMN) prosthetic arm. While NDUFA13 itself is not directly involved in this catalytic process, its structural role helps maintain the proper conformation of Complex I required for efficient electron transfer .

What experimental techniques are most effective for studying NDUFA13 expression and function in different species?

For effective characterization of NDUFA13 across species including Gorilla gorilla gorilla, researchers should consider a multi-modal approach:

• Gene expression analysis:

  • RT-qPCR with species-specific primers designed from conserved regions

  • RNA-Seq for comparative transcriptomics

  • Northern blotting for transcript size verification

• Protein detection and localization:

  • Western blotting using antibodies raised against conserved epitopes

  • Immunofluorescence microscopy (as demonstrated in studies with human NDUFA13)

  • Subcellular fractionation to confirm mitochondrial localization

• Functional characterization:

  • Oxygen consumption rate (OCR) measurements using substrate-driven respiration assays

  • Mitochondrial membrane potential assessments

  • ROS detection assays (particularly for H₂O₂ and superoxide)

• Recombinant protein studies:

  • Bacterial or baculovirus expression systems (similar to those used for other Complex I subunits)

  • Affinity purification using appropriate tags

  • In vitro reconstitution assays with other Complex I components

For species-specific studies, researchers should validate antibodies and primers using sequence alignments to ensure cross-reactivity or specificity as required.

How does moderate downregulation of NDUFA13 affect ROS generation and cellular protection mechanisms?

Research on cardiac-specific NDUFA13 heterozygous knockout (cHet) mice has revealed an intriguing relationship between NDUFA13 levels, ROS generation, and cellular protection:

• Moderate downregulation effects:

  • Creates a controlled electron leak within Complex I

  • Increases cytoplasmic H₂O₂ levels without affecting mitochondrial H₂O₂ or superoxide levels

  • Preserves oxygen consumption capacity through compensatory mechanisms

  • Activates STAT3 signaling through H₂O₂-mediated dimerization

• Protective mechanisms against ischemia-reperfusion injury:

  • Reduced apoptosis (fewer TUNEL-positive cardiomyocytes)

  • Decreased cleaved caspase-3 expression

  • Reduced cytochrome C release into cytosol

  • Smaller infarct size

• Comparative oxygen consumption rate data:

When NDUFA13 was moderately downregulated in cHet mice, the following substrate-driven oxygen consumption rates were observed:

What are the methodological considerations for generating and validating recombinant NDUFA13 from non-human primates?

When producing recombinant Gorilla gorilla gorilla NDUFA13, researchers should consider:

• Expression system selection:

  • Baculovirus systems have proven effective for other Complex I subunits

  • Mammalian expression systems may provide more appropriate post-translational modifications

  • Bacterial systems may be suitable for structural studies but might lack proper folding

• Construct design considerations:

  • Include appropriate purification tags (His, GST, etc.)

  • Consider codon optimization for the chosen expression system

  • Include proper signal sequences if mitochondrial targeting is desired

  • Design constructs with and without the hydrophobic domain for solubility testing

• Validation methods:

  • Mass spectrometry to confirm protein identity and modifications

  • Circular dichroism to assess secondary structure composition (expected to be primarily alpha-helical)

  • Functional reconstitution assays with other Complex I components

  • Integration into membrane mimetics (nanodiscs, liposomes) for transmembrane domain studies

• Comparative analysis between species:

  • Sequence alignment to identify conserved and divergent regions

  • Structural modeling based on known Complex I structures

  • Functional complementation assays in NDUFA13-deficient cell lines

How can researchers effectively investigate the relationship between NDUFA13 and STAT3 signaling in experimental models?

The NDUFA13-STAT3 relationship presents an intriguing research avenue with these methodological approaches:

• For protein-protein interaction studies:

  • Co-immunoprecipitation assays to detect physical interaction

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET approaches for real-time interaction dynamics

  • Yeast two-hybrid or mammalian two-hybrid screening to map interaction domains

• For signaling pathway analysis:

  • Phospho-specific western blotting for STAT3 activation (Tyr705, Ser727)

  • STAT3 dimerization assays using non-reducing gel electrophoresis

  • Chromatin immunoprecipitation to assess STAT3 binding to target genes

  • Luciferase reporter assays for STAT3 transcriptional activity

• For ROS-mediated STAT3 activation:

  • Subcellular-targeted ROS sensors (cytosolic vs. mitochondrial)

  • Antioxidant treatments to block H₂O₂-mediated STAT3 activation

  • Site-directed mutagenesis of redox-sensitive STAT3 residues

  • Time-course experiments to establish causality between ROS elevation and STAT3 activation

Research has demonstrated that moderate NDUFA13 downregulation increases cytosolic H₂O₂, which serves as a second messenger leading to STAT3 dimerization and activation of antiapoptotic signaling . This mechanism contributes significantly to cardioprotection during ischemia-reperfusion injury.

What approaches can be used to study the consequences of NDUFA13 mutations or variants across different species?

To investigate NDUFA13 variants across species including Gorilla gorilla gorilla:

• Comparative genomics approach:

  • Whole genome/exome sequencing to identify natural variants

  • Population genetics analysis to identify conserved vs. variable regions

  • Evolutionary rate analysis to identify regions under selection pressure

• Functional genomics strategy:

  • CRISPR/Cas9 gene editing to introduce equivalent mutations across species

  • Conditional knockout/knockdown systems (as demonstrated in cardiac-specific models)

  • Rescue experiments with wild-type and mutant variants

• Physiological assessment techniques:

  • Mitochondrial respiration assays with tissue-specific samples

  • ROS generation measurements specific to different species

  • Apoptosis assays under basal and stress conditions (e.g., hypoxia/reoxygenation)

• Structural biology considerations:

  • Cryo-EM of intact Complex I with wild-type and variant NDUFA13

  • Molecular dynamics simulations to predict mutation effects

  • In silico modeling of electron flow alterations

How should researchers design experiments to investigate the dual role of NDUFA13 in mitochondrial function and cell death regulation?

NDUFA13's dual function requires careful experimental design:

• Separation of mitochondrial and cell death functions:

  • Domain-specific mutations to disrupt specific functions

  • Subcellular targeting constructs that localize to either mitochondria or cytosol

  • Time-course experiments to establish temporal relationships

• Cell death pathway analysis:

  • Comprehensive apoptosis marker assessment (caspase-3, caspase-9, but not caspase-8)

  • Cytochrome C release quantification

  • TUNEL staining coupled with mitochondrial markers

  • Flow cytometry for early/late apoptotic populations

• Stress condition variables:

  • Hypoxia/reoxygenation protocols (6h hypoxia followed by 18h reoxygenation has been effective)

  • Ischemia/reperfusion models for in vivo studies

  • Drug-induced mitochondrial stress

  • Metabolic stress conditions

• Critical control experiments:

  • Use of both homozygous and heterozygous knockout models

  • Dose-dependent siRNA approaches (100 μmol/L vs. 200 μmol/L show different effects)

  • Osmotic controls (mannitol) for high glucose experiments

  • Monitoring of mitochondrial membrane potential alongside functional assays

Research has shown that moderate NDUFA13 downregulation (as in heterozygous knockout or low-dose siRNA) offers protection against apoptosis, while severe downregulation (homozygous knockout or high-dose siRNA) may impair mitochondrial function without protective benefits .

What are the optimal conditions for expressing and purifying recombinant NDUFA13 while maintaining its structural integrity?

For successful recombinant NDUFA13 production:

• Expression system optimization:

  • Baculovirus systems have proven effective for Complex I subunits

  • Consider using insect cells (Sf9, High Five) for eukaryotic processing

  • Evaluate bacterial systems with solubility-enhancing fusion partners

  • Implement temperature and induction optimization protocols

• Purification strategy:

  • Two-step purification combining affinity and size exclusion chromatography

  • Detergent selection critical for membrane domain solubilization

  • Consider mild detergents (DDM, LMNG) over harsh detergents (SDS, Triton X-100)

  • Maintain reducing conditions throughout purification

• Stability considerations:

  • Buffer optimization with varied pH (7.0-8.0) and salt concentrations

  • Addition of glycerol (5-10%) to enhance stability

  • Consider lipid supplementation for transmembrane domain stability

  • Implement thermal shift assays to assess proper folding

• Quality control measures:

  • SEC-MALS to confirm monomeric state and molecular weight

  • Negative stain electron microscopy for structural integrity

  • Functional assays to confirm activity if combined with other Complex I components

  • Storage stability tests at different temperatures

How can researchers effectively distinguish between the direct effects of NDUFA13 modulation and secondary consequences in complex experimental systems?

To establish causality in NDUFA13 studies:

• Genetic approaches with appropriate controls:

  • Use of inducible systems with precise temporal control

  • Rescue experiments with wild-type and mutant constructs

  • Implementation of domain-specific mutations

  • Generation of dose-dependent knockdown models

• Pharmacological intervention strategy:

  • ROS scavengers to block H₂O₂-mediated effects

  • STAT3 inhibitors to block downstream signaling

  • Complex I inhibitors (rotenone) as positive controls

  • Mitochondrial-targeted vs. cytosolic antioxidants to distinguish compartment-specific effects

• Time-course experimental design:

  • Early vs. late timepoints to establish sequential events

  • Pulse-chase approaches to track specific molecular changes

  • Real-time monitoring where possible

• Multiparameter analysis:

  • Simultaneous assessment of multiple endpoints:

This comprehensive approach helps distinguish primary from secondary effects of NDUFA13 modulation .

How should researchers interpret conflicting data regarding NDUFA13's role in apoptosis across different experimental models?

Resolving conflicting findings requires systematic analysis:

• Context-dependent factors to consider:

  • Cell/tissue type specificity (cardiac vs. tumor cells show different responses)

  • Level of NDUFA13 reduction (moderate vs. severe downregulation)

  • Acute vs. chronic modulation

  • Basal state vs. stress conditions (particularly ischemia/reperfusion)

• Mechanistic reconciliation approaches:

  • Distinguish intrinsic vs. extrinsic apoptosis pathways

  • Separate mitochondrial function from cell death regulation

  • Consider threshold effects in ROS signaling

  • Evaluate compensatory mechanisms in different models

• Systematic analysis framework:

  • Compare experimental conditions across studies

  • Evaluate species differences

  • Assess temporal dynamics of responses

  • Consider interaction with other signaling pathways

• Key data reconciliation insights:

  • Moderate NDUFA13 reduction protects against apoptosis by activating STAT3 through increased cytosolic H₂O₂

  • Severe NDUFA13 reduction impairs mitochondrial function without protective benefits

  • Tumor cells may have different baseline STAT3 activation status

  • Cell-specific responses may reflect different metabolic dependencies

What statistical approaches are most appropriate for analyzing the complex datasets generated in NDUFA13 research?

For robust statistical analysis in NDUFA13 studies:

• Experimental design considerations:

  • Power analysis to determine appropriate sample sizes

  • Factorial designs to evaluate multiple variables simultaneously

  • Repeated measures designs for time-course experiments

  • Hierarchical designs for nested data (e.g., animals → tissues → cells)

• Primary statistical approaches:

  • ANOVA with appropriate post-hoc tests for multiple group comparisons

  • Mixed-effects models for complex experimental designs

  • Survival analysis for time-to-event data (e.g., Kaplan-Meier curves)

  • Multivariate analyses for correlated outcomes

• Advanced analytical methods:

  • Principal component analysis for dimensionality reduction

  • Cluster analysis to identify patterns in complex datasets

  • Pathway enrichment analysis for transcriptomic/proteomic data

  • Meta-analysis approaches to integrate findings across studies

• Reporting standards:

  • Effect sizes alongside p-values

  • Confidence intervals for key measurements

  • Transparent reporting of exclusion criteria

  • Validation in independent datasets where possible

In published research, statistical significance between NDUFA13 heterozygous knockout and control mice was established using appropriate statistical tests, with p-values reported for various parameters including cardiac function, apoptosis markers, and mitochondrial function measures .