Recombinant Macaca fascicularis NADH-ubiquinone oxidoreductase chain 5 (MT-ND5)

What is the normal function of MT-ND5 in cellular metabolism?

MT-ND5 encodes NADH dehydrogenase 5, a core subunit of mitochondrial Complex I involved in oxidative phosphorylation. This protein participates in the first step of the electron transport process, transferring electrons from NADH to ubiquinone. Within mitochondria, Complex I is embedded in the inner mitochondrial membrane where it contributes to creating an unequal electrical charge through the step-by-step transfer of electrons. This electrochemical gradient is essential for ATP production, the cell's primary energy source. MT-ND5 is therefore crucial for cellular energy metabolism, converting food energy into a usable form for cells through the process of oxidative phosphorylation.

How does MT-ND5 from Macaca fascicularis differ from human MT-ND5?

Macaca fascicularis (crab-eating macaque) MT-ND5 shares significant sequence homology with human MT-ND5, making it a valuable model for human mitochondrial research. The Macaca fascicularis MT-ND5 protein (Uniprot P50665) functions similarly to its human counterpart as part of Complex I in the respiratory chain. The amino acid sequence of the recombinant form includes: "MIMHTPIMMTTLISLTLPIFATLTNPYKKRSYPDYVKTTVMYAFITSLPSTTLFILSNQETTIWSWHWMTTQTLDLTLS," representing positions 1-79 of the protein. Conservation analysis shows that key functional domains are preserved between species, though species-specific variations exist that may affect protein-protein interactions within the complex or subtle differences in electron transfer efficiency.

What are the common alternative names and identifiers for MT-ND5 in scientific literature?

When conducting literature searches or database queries related to MT-ND5, researchers should be aware of various nomenclature used across publications. MT-ND5 is also known as:

• NADH-ubiquinone oxidoreductase chain 5 (official recommended name)

• NADH dehydrogenase subunit 5 (alternative name)

• MTND5 (previous HGNC symbol)

• NADH5 (synonym)

• ND5 (synonym)

Important database identifiers include:

• Uniprot: P50665 (Macaca fascicularis), P03915 (Human)

• HGNC: 7461 (Human)

• NCBI Gene: 4540 (Human)

• Ensembl: ENSG00000198786 (Human)

• OMIM: 516005 (Human)

How is MT-ND5 expression regulated in different tissues?

MT-ND5 expression patterns vary across tissues based on energy demands, with highest expression in tissues requiring substantial ATP production. As a mitochondrially-encoded gene, MT-ND5 is regulated differently from nuclear genes. Its expression is influenced by mitochondrial DNA copy number, which can vary from hundreds to thousands per cell depending on tissue type. Brain tissue, which consumes >20% of the body's energy despite constituting only ~2% of body weight, shows particularly high expression. Studies in mouse models demonstrate that organs with high oxygen consumption are especially dependent on proper MT-ND5 function. This tissue-specific expression pattern correlates with sensitivity to MT-ND5 mutations, explaining why tissues like brain, heart, and skeletal muscle are most affected in mitochondrial disorders.

What are the optimal storage and handling conditions for recombinant Macaca fascicularis MT-ND5?

For maintaining protein stability and activity, recombinant Macaca fascicularis MT-ND5 requires specific storage conditions. The protein is typically supplied in a Tris-based buffer with 50% glycerol, optimized for stability. For long-term storage, the protein should be kept at -20°C, with extended storage recommended at -80°C to prevent degradation. To minimize freeze-thaw cycles that can compromise protein integrity, working aliquots should be prepared and stored at 4°C for up to one week. Repeated freezing and thawing significantly reduces protein activity and should be avoided. For experimental applications, researchers should maintain the protein's optimal pH and ionic conditions specific to the assay being performed.

How can researchers assess MT-ND5 functionality in experimental models?

Functional assessment of MT-ND5 requires multiple complementary approaches:

• Oxygen Consumption Rate (OCR) Measurement: Using platforms like Seahorse XF Analyzer to quantify mitochondrial respiration. In MT-ND5 mutant mouse embryonic fibroblasts (MEFs), significantly lower OCR has been observed compared to wild-type cells, directly demonstrating impaired ATP synthesis.

• ATP Synthesis Quantification: Direct measurement of ATP production in tissues or cell cultures can reveal MT-ND5 functionality. Research shows that tissues from MT-ND5 knockout mice exhibit substantially reduced ATP synthesis compared to wild-type specimens.

• Electron Transport Chain Complex Activity Assays: Specific enzymatic assays measuring NADH:ubiquinone oxidoreductase activity can assess Complex I function.

• Mitochondrial Membrane Potential Analysis: Using fluorescent probes to evaluate the electrochemical gradient that MT-ND5 helps establish.

• Transmission Electron Microscopy: To visualize structural changes in mitochondrial cristae, as damaged cristae have been observed in MT-ND5 mutant mice.

What methodological approaches are recommended for studying MT-ND5 mutations?

When investigating MT-ND5 mutations, researchers should employ a multi-faceted approach:

• Heteroplasmy Quantification: As MT-ND5 is encoded in mitochondrial DNA, determining the proportion of mutant to wild-type mtDNA is crucial. Digital droplet PCR or next-generation sequencing can provide accurate heteroplasmy measurements.

• Functional Consequences Assessment: Measuring Complex I activity, oxygen consumption rates, and ATP production in cells harboring mutations.

• Phenotypic Analysis: In animal models, comprehensive phenotyping including metabolic parameters, thermoregulation ability, and tissue-specific effects. For example, MT-ND5 knockout mice show decreased oxygen consumption and CO₂ production rates during the dark cycle, alongside impaired heat production and abnormal drinking behavior.

• Off-Target Effect Evaluation: Whole-mitochondrial genome sequencing to identify potential off-target effects, as shown in studies where MT-ND5 nonsense mutant mice presented several off-target activities at multiple mtDNA loci with varying editing efficiencies.

• Biochemical and Genetic Testing: Essential for accurate diagnosis, particularly in cases where MT-ND5 disorders may be misdiagnosed as other conditions.

How can researchers effectively design experiments to study MT-ND5 in complex disease models?

Designing robust experiments for studying MT-ND5 in disease contexts requires careful consideration of several factors:

• Model Selection: Choose models that recapitulate human disease phenotypes. Both cell-based systems (patient-derived fibroblasts, cybrid cells) and animal models (transgenic mice) have proven valuable. Recent studies have established MEF cell lines from MT-ND5 knockout mice to assess oxygen consumption rates and validate mitochondrial dysfunction.

• Controls for Mitochondrial Heteroplasmy: Include appropriate controls accounting for the heteroplasmic nature of mtDNA mutations. Isogenic controls with matched nuclear backgrounds but different mitochondrial genotypes are ideal.

• Tissue-Specific Effects: Design experiments to examine tissue-specific consequences, particularly in high-energy tissues (brain, heart, muscle). Studies have demonstrated that despite being a systemic genetic defect, MT-ND5 mutations manifest differently across tissues.

• Temporal Considerations: Include time-course analyses to capture progressive phenotypes, as mitochondrial dysfunction often worsens over time.

• Environmental Challenges: Incorporate stressors that unmask subtle phenotypes. For example, cold exposure (6°C) revealed significant thermoregulation defects in MT-ND5 mutant mice that weren't apparent under standard conditions.

• Multi-omics Approach: Combine transcriptomics, proteomics, and metabolomics for comprehensive characterization of molecular changes.

What disease phenotypes are associated with MT-ND5 mutations?

MT-ND5 mutations are associated with several distinct clinical phenotypes:

• Leigh Syndrome: A severe neurological disorder characterized by vomiting, seizures, delayed development, muscle weakness, and movement problems. Some children with MT-ND5-related Leigh syndrome exhibit atypical features including intrauterine growth retardation. The condition often affects the brainstem and basal ganglia, and can progress rapidly.

• MELAS Syndrome (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes): Characterized by stroke-like episodes, encephalopathy with seizures and dementia, and lactic acidosis. MT-ND5 has been specifically implicated in this syndrome.

• Leber Hereditary Optic Neuropathy (LHON): MT-ND5 acts as a modifier gene for this condition, which causes sudden-onset bilateral vision loss primarily affecting young adult males.

• Metabolic Disorders: MT-ND5 knockout models demonstrate obesity phenotypes with significantly larger white adipose tissues and brown adipose tissues compared to wild-type specimens, indicating a role in metabolic regulation.

• Thermoregulation Defects: MT-ND5 mutant mice show decreased heat production ability and abnormal thermoregulation, particularly evident under cold stress conditions.

How can researchers differentiate MT-ND5-related mitochondrial disorders from similar conditions?

Distinguishing MT-ND5-related disorders from phenotypically similar conditions requires a systematic approach:

• Biochemical Testing: Measure lactate levels, pyruvate levels, and lactate-to-pyruvate ratios in blood and cerebrospinal fluid. MT-ND5 disorders typically show elevated lactate due to impaired oxidative phosphorylation.

• Complex I Activity Assays: Directly measure Complex I function in muscle biopsies or cultured fibroblasts. MT-ND5 mutations specifically impair Complex I activity while preserving other respiratory chain complexes.

• Genetic Analysis: Perform mitochondrial DNA sequencing focused on MT-ND5. Next-generation sequencing allows detection of low-level heteroplasmy that might be missed by Sanger sequencing.

• Imaging Studies: MRI patterns in MT-ND5-related disorders often show specific distributions of lesions, particularly in Leigh syndrome and MELAS.

• Tissue Biopsy Analysis: Histochemical and electron microscopy examination of muscle biopsies can reveal ragged red fibers and abnormal mitochondria characteristic of mitochondrial diseases.

Case studies have documented instances where MT-ND5-related mitochondrial disorders were initially misdiagnosed as other conditions, emphasizing the importance of comprehensive biochemical and genetic testing in atypical presentations.

What are the experimental challenges in modeling MT-ND5-related diseases?

Researchers face several unique challenges when modeling MT-ND5-related diseases:

• Heteroplasmy Management: mtDNA mutations exist in varying proportions (heteroplasmy) within cells and tissues, creating experimental variability. Research models must control for heteroplasmy levels to ensure reproducible results.

• Threshold Effects: Phenotypes may only manifest when mutation loads exceed tissue-specific thresholds, requiring careful quantification of mutation burden.

• Nuclear-Mitochondrial Interactions: The nuclear genetic background significantly influences how MT-ND5 mutations manifest, necessitating controlled genetic backgrounds in experimental models.

• Tissue Specificity: Despite the mutation being present in all cells, disease manifestations vary dramatically across tissues. Comprehensive multi-tissue analysis is required, as demonstrated in studies examining brain, adipose tissue, and other organs in MT-ND5 knockout mice.

• Off-Target Effects: In gene editing approaches targeting MT-ND5, researchers must account for potential off-target effects. Whole-mitochondrial genome sequencing has revealed off-target activities at multiple mtDNA loci with varying editing efficiencies in MT-ND5 mutant mice.

• Progressive Nature: The progressive nature of mitochondrial diseases requires longitudinal studies over extended timeframes to capture the full disease spectrum.

How do MT-ND5 mutations affect cellular metabolism beyond ATP production?

MT-ND5 mutations impact cellular metabolism through multiple interconnected pathways beyond direct ATP production:

• Reactive Oxygen Species (ROS) Production: Dysfunctional Complex I can increase ROS generation, triggering oxidative stress and damage to proteins, lipids, and DNA. Specific MT-ND5 variants (m.12372G>A) have been associated with lower ROS production rates.

• Calcium Homeostasis: Mitochondrial membrane potential disruption affects calcium signaling and storage, impacting processes from neurotransmission to muscle contraction.

• Metabolic Remodeling: Cells with MT-ND5 mutations often exhibit compensatory metabolic shifts, including increased glycolysis and altered amino acid metabolism. These shifts represent attempts to maintain energy production through alternative pathways.

• Thermoregulation: MT-ND5 knockout mice demonstrate significant impairment in heat production. During cold challenge experiments (6°C), mutant mice showed progressive decreases in rectal temperature and reduced survival compared to wild-type mice.

• Adipose Tissue Metabolism: MT-ND5 knockout mice develop an obesity phenotype with significantly enlarged white and brown adipose tissues. Brown adipocytes from mutant mice were approximately 1.8 times larger than those from wild-type mice, with visibly damaged mitochondrial cristae observed using transmission electron microscopy.

• Glucose Metabolism: Altered glucose handling has been observed in MT-ND5 mutant models, suggesting broader impacts on whole-body metabolism and potentially contributing to metabolic disease phenotypes.

How can Macaca fascicularis MT-ND5 serve as a model for human MT-ND5 research?

Macaca fascicularis MT-ND5 provides several advantages as a model for human mitochondrial research:

• Evolutionary Conservation: Significant sequence and functional conservation exists between human and macaque MT-ND5, with critical functional domains preserved. This conservation allows findings in macaque models to be reasonably extrapolated to human biology.

• Translational Research Pipeline: As non-human primates, Macaca fascicularis offers a closer physiological match to humans than rodent models, particularly for studies of complex neurological and metabolic conditions associated with MT-ND5 dysfunction.

• Recombinant Protein Availability: The availability of recombinant Macaca fascicularis MT-ND5 protein enables detailed biochemical studies of protein function, enzyme kinetics, and structural analyses without the complexities of whole-organism models.

• Cross-Species Validation: Findings from rodent models can be validated in macaque systems as an intermediate step before human studies, strengthening translational research pipelines in mitochondrial biology.

• System-Level Integration: The similar organization of metabolic networks between primates allows for more accurate modeling of how MT-ND5 perturbations affect integrated physiological systems.

For researchers, these advantages must be balanced against the ethical and practical considerations of non-human primate research, with appropriate experimental design to maximize scientific value while minimizing animal usage.

What are the key structural and functional differences between MT-ND5 across mammalian species?

Comparative analysis of MT-ND5 across mammals reveals important evolutionary patterns:

• Sequence Variation: While core catalytic domains show high conservation, species-specific variations occur particularly in regions involved in assembly and supramolecular interactions. The amino acid sequence of Macaca fascicularis MT-ND5 (positions 1-79: "MIMHTPIMMTTLISLTLPIFATLTNPYKKRSYPDYVKTTVMYAFITSLPSTTLFILSNQETTIWSWHWMTTQTLDLTLS") shows characteristic primate features.

• Thermal Adaptation: Species adapted to different thermal environments show variations in MT-ND5 that may affect the temperature sensitivity of Complex I function. This is particularly relevant given the observed thermoregulation defects in MT-ND5 knockout mice.

• Metabolic Rate Correlation: MT-ND5 variations correlate with basal metabolic rates across species, with sequence changes potentially optimizing energy production efficiency for species-specific metabolic demands.

• ROS Production Differences: Variations in MT-ND5 across species affect the propensity for reactive oxygen species generation, with implications for species differences in aging and oxidative stress responses.

• Disease Susceptibility: Certain pathogenic mutations in human MT-ND5 align with normal sequence variations in other species, suggesting compensatory mechanisms exist in those species that might inform therapeutic approaches.

These differences must be considered when extrapolating findings across species, particularly when using animal models to study human disease or when developing therapies targeting Complex I function.

How do researchers control for species-specific variables when using Macaca fascicularis MT-ND5 in comparative studies?

When conducting comparative studies with Macaca fascicularis MT-ND5, researchers must address several species-specific variables:

• Background Genetic Variation: Control for nuclear genome differences that may influence mitochondrial function through nuclear-encoded mitochondrial proteins that interact with MT-ND5.

• Experimental System Selection: Choose appropriate experimental systems (isolated proteins, cell cultures, tissue samples) based on the specific research question, recognizing that some aspects of MT-ND5 function may be system-dependent.

• Physiological Parameter Adjustment: Account for baseline differences in physiological parameters between species (body temperature, metabolic rate, lifespan) when interpreting experimental results.

• Environmental Standardization: Maintain consistent environmental conditions across experiments, as MT-ND5 function can be influenced by temperature, nutrient availability, and other environmental factors.

• Multi-Species Validation: When possible, validate key findings across multiple species to distinguish conserved functions from species-specific adaptations.

• Bioinformatic Approaches: Employ computational analyses to identify conserved domains versus variable regions in MT-ND5, focusing experimental work on regions most likely to yield translatable results.

• Heterologous Expression Systems: Use defined expression systems where MT-ND5 variants can be studied against controlled backgrounds to isolate species-specific effects.

What experimental designs best capture the impact of MT-ND5 mutations on mitochondrial function?

Optimal experimental designs for studying MT-ND5 mutation effects include:

• Cybrid Cell Models: Transferring mitochondria from cells carrying MT-ND5 mutations into mtDNA-depleted recipient cells creates cybrid lines with identical nuclear backgrounds but different mitochondrial genotypes. This isolates the effects of MT-ND5 mutations from confounding nuclear genetic factors.

• Tissue-Specific Conditional Knockout Models: Since MT-ND5 mutations affect tissues differently, conditional knockout models allow researchers to study tissue-specific effects. Studies have demonstrated that high-energy tissues like brain and brown adipose tissue show pronounced effects from MT-ND5 dysfunction.

• Heteroplasmy Titration Experiments: Creating cell lines with controlled proportions of mutant mtDNA allows determination of threshold effects—the percentage of mutant mtDNA required to manifest biochemical and physiological defects.

• Challenge Testing Protocols: Exposing models to stressors that increase energy demands or challenge mitochondrial function can unmask subtle phenotypes. Cold challenge experiments (6°C exposure) revealed significant thermoregulation defects in MT-ND5 mutant mice that weren't apparent under standard conditions.

• Multi-Parameter Phenotyping: Comprehensive assessment across multiple parameters, including:

  • Oxygen consumption and CO₂ production rates

  • ATP synthesis efficiency

  • Heat production capacity

  • Morphological analysis of mitochondria using transmission electron microscopy

  • Metabolic parameters (glucose levels, adipose tissue characteristics)

What controls should be included when designing MT-ND5 knockout or mutation studies?

Robust MT-ND5 studies require comprehensive controls:

• Isogenic Controls: Use cell lines or animals with identical nuclear backgrounds to isolate mitochondrial effects.

• Heteroplasmy Controls: Include samples with varying levels of mutation load to establish threshold effects, as phenotype severity often correlates with heteroplasmy percentage.

• Tissue-Matched Controls: Since MT-ND5 effects vary by tissue, controls should be matched for tissue type and, when possible, for developmental stage and environmental exposure history.

• Rescue Controls: Complementation with wild-type MT-ND5 or alternative energy pathway activation to demonstrate specificity of observed effects.

• Off-Target Effect Controls: Whole-mitochondrial genome sequencing to identify potential off-target activities, as MT-ND5 mutant mice have shown off-target effects at multiple mtDNA loci with varying editing efficiencies.

• Environmental Controls: Standardize environmental conditions (temperature, feeding, activity cycles) as these significantly impact mitochondrial function and can confound results if not controlled.

• Age and Sex-Matched Controls: Mitochondrial function changes with age and differs between sexes, making these important variables to control.

• Technical Controls: Include measurements of multiple respiratory chain complexes to distinguish MT-ND5-specific effects (Complex I) from general mitochondrial dysfunction.

What are the most sensitive assays for detecting subtle changes in MT-ND5 function?

Detection of subtle MT-ND5 functional changes requires highly sensitive methodologies:

• High-Resolution Respirometry: Platforms like Oroboros O2k or Seahorse XF analyzers can detect minor changes in oxygen consumption rates and calculate respiratory control ratios that reveal coupling efficiency alterations.

• In-Gel Activity Assays: Blue Native PAGE coupled with in-gel activity staining provides visual and quantitative assessment of assembled Complex I activity.

• Supercomplex Assembly Analysis: As MT-ND5 affects supercomplex formation, techniques that preserve and analyze supercomplex structures (clear native PAGE, cryo-electron microscopy) can detect subtle assembly defects.

• Redox State Monitoring: Real-time fluorescent monitoring of NAD+/NADH ratios provides dynamic readouts of electron transport efficiency.

• Mitochondrial Membrane Potential Dynamics: Time-resolved measurements of membrane potential fluctuations using voltage-sensitive dyes can detect subtle bioenergetic changes before steady-state values are affected.

• Metabolic Flux Analysis: Isotope tracing combined with mass spectrometry to track metabolic pathway usage and identify compensatory mechanisms activated by subtle MT-ND5 dysfunction.

• Single-Cell Analysis: Technologies that measure mitochondrial function in individual cells can detect heterogeneous responses that might be masked in population averages, particularly important given the heteroplasmic nature of MT-ND5 mutations.

How can researchers effectively isolate and purify recombinant MT-ND5 for structural and functional studies?

Purification of recombinant MT-ND5 requires specialized approaches due to its hydrophobic nature and complex structural characteristics:

• Expression System Selection: Mammalian expression systems often yield better results than bacterial systems for mitochondrial membrane proteins. For Macaca fascicularis MT-ND5, mammalian cell lines transfected with optimized expression vectors provide good balance between yield and proper folding.

• Solubilization Strategy: Using appropriate detergents is critical—mild non-ionic detergents (DDM, LMNG) or styrene maleic acid copolymers (SMALPs) preserve structural integrity better than harsh ionic detergents.

• Affinity Purification: Incorporation of affinity tags (His, FLAG, Strep) facilitates purification, though tag placement must be carefully considered to avoid interfering with protein folding or function. The tag type for recombinant Macaca fascicularis MT-ND5 is typically determined during the production process to optimize protein yield and activity.

• Stability Enhancement: Addition of specific lipids during purification helps maintain native-like environment and structural stability.

• Quality Control Metrics: Employ multiple quality control steps including:

  • Size-exclusion chromatography to assess aggregation state

  • Activity assays to confirm functionality

  • Circular dichroism to verify secondary structure

  • Thermal shift assays to determine stability

• Storage Considerations: Store in appropriate buffer conditions with 50% glycerol at -20°C for regular use or -80°C for extended storage. Avoid repeated freeze-thaw cycles that significantly reduce protein activity.

• Complex Assembly: For certain studies, co-expression with other Complex I components may yield more stable and functionally relevant preparations than isolated MT-ND5.