Recombinant Sigmodon hispidus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)
Description
Protein Overview
MT-ND3 is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase, commonly known as Complex I. Located in the mitochondrial inner membrane, Complex I represents the largest of the five respiratory complexes and catalyzes electron transfer from NADH to ubiquinone . The protein is encoded by the mitochondrial genome rather than the nuclear genome, which has significant implications for its inheritance pattern and role in mitochondrial diseases.
The recombinant version of Sigmodon hispidus MT-ND3 is produced through advanced protein expression systems to create a functional protein that mirrors the native form found in the Hispid cotton rat. This recombinant protein serves as a valuable research tool for investigating mitochondrial function and related disorders.
Comparison with Other Species
Recombinant MT-ND3 proteins have been produced from several Sigmodon species, allowing for comparative studies. The table below compares key characteristics of MT-ND3 from different sources:
The conservation of MT-ND3 across species highlights its evolutionary importance in mitochondrial function, while species-specific variations provide insights into adaptive differences in energy metabolism.
Role in Complex I
NADH-ubiquinone oxidoreductase (Complex I) is the first and largest enzyme complex in the mitochondrial respiratory chain. MT-ND3 serves as a critical component of this complex, contributing to its structural integrity and functional capability . The complex catalyzes the transfer of electrons from NADH to ubiquinone, coupled with proton translocation across the inner mitochondrial membrane, which is essential for ATP production through oxidative phosphorylation.
As one of the most hydrophobic subunits of Complex I, MT-ND3 is integral to forming the core of the transmembrane region . This positioning enables it to participate in the proton pumping mechanism that establishes the electrochemical gradient necessary for ATP synthesis.
Regulatory Mechanisms
Interestingly, research has identified MT-ND3 as a target of thyroid hormone regulation. Studies have confirmed the presence of a thyroid hormone receptor (TR)/c-erbA specific binding site in the mitochondrial ND3 gene . This finding suggests direct transcriptional regulation of this mitochondrial gene by thyroid hormone.
In rat models, hypothyroidism has been shown to decrease ND3 mRNA levels in several brain areas, including the cortex and hippocampus, particularly during early postnatal development . This hormonal regulation indicates an additional layer of control over mitochondrial energy production that may be particularly important during developmental stages.
Laboratory Applications
Recombinant Sigmodon hispidus MT-ND3 has numerous applications in mitochondrial research, including:
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Structure-function studies of Complex I
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Investigation of mitochondrial disorders
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Drug screening for compounds that modulate Complex I activity
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Comparative studies of mitochondrial function across species
MT-ND3 Variants and Mitochondrial Diseases
Mutations in the MT-ND3 gene have been associated with several mitochondrial disorders. Pathogenic variants are known to cause mitochondrial complex I deficiency (MT-C1D) and may lead to various clinical manifestations, including Leigh syndrome, Leber hereditary optic neuropathy, and encephalopathy .
Recent research has identified a novel m.10197G > C variant in MT-ND3 in a patient with mitochondrial disease, as well as two other patients with m.10191T > C variants . Functional analyses of the novel m.10197G > C variant revealed that it significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, along with reduction of ATP synthesis .
Therapeutic Approaches and Research Breakthroughs
A promising approach for addressing MT-ND3-related mitochondrial disorders involves re-engineering techniques to deliver mitochondrial genes into mitochondria. This is accomplished through codon optimization for nuclear expression and translation by cytoplasmic ribosomes, followed by import into mitochondria .
In a groundbreaking study, researchers constructed mitochondrial targeting sequences along with codon-optimized MT-ND3 and successfully imported them into mitochondria of patients with m.10197G > C and m.10191T > C missense variants in MT-ND3 . This nuclear expression of the MT-ND3 gene partially restored protein levels, improved complex I deficiency, and significantly enhanced ATP production, indicating a functional rescue of the mutant phenotype .
These findings suggest that codon-optimized nuclear expression of mitochondrial proteins and their import into mitochondria could supplement ATP requirements in energy-deficient mitochondrial disease patients, opening new therapeutic avenues for these challenging conditions.
Detection and Quantification Techniques
Several methods have been developed to study MT-ND3 and Complex I activity. These include:
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Western Blotting: Using specific antibodies such as MT-ND3 (E8O4E) Rabbit mAb, which can be used at a dilution of 1:1000 for detecting the approximately 13 kDa MT-ND3 protein .
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Immunoprecipitation: Antibodies against MT-ND3 can be used at a dilution of 1:200 for immunoprecipitation studies .
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In vivo Autoradiographic Assay: An autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding after intravenous administration has been developed to assess Complex I (which includes MT-ND3) in the living brain . This technique shows regional heterogeneity in binding, with enrichment in neurons compared to glia, and the lowest levels in white matter .
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ELISA: Enzyme-linked immunosorbent assays using recombinant MT-ND3 proteins are available for quantitative assessment of protein levels and interactions .
Correlation with Other Mitochondrial Enzymes
Research has demonstrated an excellent correlation between regional levels of in vivo [(3)H]DHR binding (reflecting Complex I activity) and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase) . This correlation suggests that the stoichiometry of these components of the electron transport chain remains relatively constant across different brain regions, providing valuable insights into the organization and regulation of mitochondrial respiration.
Emerging Therapeutic Strategies
The successful demonstration of importing codon-optimized MT-ND3 into mitochondria represents a significant advancement in potential therapeutic approaches for mitochondrial diseases . This allotopic expression strategy could be refined and extended to other mitochondrially encoded proteins, potentially addressing a broader spectrum of mitochondrial disorders.
Imaging Technologies
The development of in vivo assays for Complex I activity suggests potential imaging techniques for assessing mitochondrial function in humans . Further refinement of these techniques could provide valuable diagnostic tools for mitochondrial disorders and neurodegenerative diseases with mitochondrial involvement.
Comparative Studies
Continued investigation of MT-ND3 across different species, including the recombinant proteins from Sigmodon hispidus and Sigmodon ochrognathus, will provide deeper insights into the evolution of mitochondrial function and species-specific adaptations in energy metabolism.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have a specific format preference, please indicate your requirement in the order notes. We will fulfill your request whenever possible.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please contact us in advance. Additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is MT-ND3 and what is its function in mitochondrial biology?
MT-ND3 (Mitochondrial NADH-ubiquinone oxidoreductase chain 3) functions as a core subunit of mitochondrial respiratory chain complex I. This complex catalyzes electron transfer from NADH through the respiratory chain using ubiquinone as an electron acceptor . The protein is essential for complex I catalytic activity, playing a crucial role in cellular energy production through oxidative phosphorylation .
MT-ND3 is encoded by mitochondrial DNA (mtDNA) rather than nuclear DNA, making it subject to maternal inheritance patterns and potentially different mutation rates than nuclear genes . Mutations in MT-ND3 can lead to mitochondrial disorders, including Leigh syndrome and mitochondrial complex I deficiency .
How can I verify the purity and integrity of recombinant MT-ND3 protein?
Verification of recombinant MT-ND3 can be conducted through multiple complementary methods:
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SDS-PAGE analysis: Greater than 90% purity can be confirmed using this technique . Run the protein alongside molecular weight markers to verify the expected size (approximately 13 kDa for MT-ND3).
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Western blotting: Using a specific antibody against MT-ND3 to confirm identity. Commercial antibodies like those from Abcam can be used for detection .
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Mass spectrometry: For precise confirmation of the amino acid sequence and post-translational modifications.
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Functional assays: Measuring NADH dehydrogenase activity using spectrophotometric methods that track electron transfer.
When working with lyophilized protein, proper reconstitution is essential. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add glycerol (5-50% final concentration) for long-term storage stability .
What experimental conditions are optimal for handling recombinant MT-ND3?
Based on handling recommendations for similar recombinant proteins:
| Parameter | Optimal Condition | Notes |
|---|---|---|
| Storage temperature | -20°C to -80°C | Aliquoting is necessary to avoid freeze-thaw cycles |
| Working temperature | 4°C | For up to one week |
| Storage buffer | Tris/PBS-based, pH 8.0 | With 6% trehalose as stabilizer |
| Reconstitution medium | Deionized sterile water | To 0.1-1.0 mg/mL |
| Glycerol concentration | 50% | For long-term storage stability |
Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity . After reconstitution, store working aliquots at 4°C for up to one week, while maintaining long-term stocks at -20°C or preferably -80°C .
How can I design experiments to study MT-ND3 mutations in Sigmodon hispidus using molecular techniques?
When designing experiments to study MT-ND3 mutations in S. hispidus, several molecular approaches can be employed:
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Restriction site analysis: This technique has proven effective for analyzing mtDNA variation in cotton rats. A microgeographic study of 134 cotton rats revealed significant spatial and temporal heterogeneity in mtDNA genotypes, which could be applied to studying MT-ND3 variants specifically .
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ARMS-PCR (Amplification Refractory Mutation System): This method has been successfully used for quantifying mutation rates in MT-ND3 mRNA. Design specific primers that selectively amplify either wild-type or mutant sequences .
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Next-Generation Sequencing (NGS): For comprehensive detection of all variants in the MT-ND3 gene, including low-frequency heteroplasmy .
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Mitochondrial isolation protocol:
When designing primers for mutation detection, optimize primer sets by testing them on plasmids encoding target wild-type and mutant genes mixed at varying ratios (0–100%) to establish a standard curve and validate quantification accuracy .
What are the challenges in expressing recombinant mitochondrial proteins like MT-ND3, and how can these be overcome?
Expressing functional recombinant mitochondrial proteins presents several challenges:
| Challenge | Solution Strategy |
|---|---|
| Hydrophobicity | Use specialized expression vectors with solubilization tags (His, GST, MBP) |
| Codon usage bias | Optimize codons for E. coli or use Rosetta strains with rare tRNAs |
| Proper folding | Express at lower temperatures (16-25°C) with chaperone co-expression |
| Start codon variations | Modify non-ATG start codons to ATG for efficient translation in E. coli |
| Post-translational modifications | Consider eukaryotic expression systems when modifications are critical |
For MT-ND3 specifically, several considerations should be noted:
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Start codon adaptation: The natural MT-ND3 in some species lacks an ATG start codon, which can affect expression efficiency in heterologous systems. Modifying the start codon to ATG (as seen in therapeutic applications) can enhance expression .
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E. coli expression system: Has been successfully used for recombinant MT-ND3 protein with N-terminal His tags . Expression in E. coli can yield full-length protein (1-116 amino acids) with greater than 90% purity.
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Handling polyA tails: For mRNA studies, considering the role of polyA in translation is important. Artificial polyA modification may be necessary when working with exogenous mRNA delivered to mitochondria .
How can I design functional assays to evaluate the activity of recombinant MT-ND3 in complex I?
Functional assessment of recombinant MT-ND3 in complex I requires both direct and indirect approaches:
A comprehensive evaluation protocol might include:
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Baseline measurement of respiratory capacity
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Measurement of complex I-specific respiration using appropriate substrates
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Inhibitor studies using rotenone to confirm complex I-specific effects
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Comparison with control samples to determine functional rescue
What methods can be used to deliver recombinant MT-ND3 or its mRNA to mitochondria for functional studies?
Several approaches have been developed for mitochondrial delivery of proteins or mRNA:
When using the MITO-Porter system for mRNA delivery, the protocol involves:
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Preparation of the therapeutic mRNA
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Formation of liposomes encapsulating the mRNA
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Transfection into cells
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Verification of mitochondrial localization using appropriate markers
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Functional assessment of the delivered mRNA through expression and activity measurements
Dose-dependent effects have been observed, with higher doses (60 ng/well vs. 30 ng/well) resulting in greater reduction of mutation rates (10% vs. 20% compared to 80% in non-treated cells) .
How can I quantitatively assess MT-ND3 heteroplasmy levels in mitochondrial disease models?
Accurate quantification of MT-ND3 heteroplasmy is critical for understanding mitochondrial disease pathology:
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ARMS-qPCR methodology: This approach has been validated for quantifying mutation rates in MT-ND3 mRNA:
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Design allele-specific primers that selectively amplify either wild-type or mutant sequences
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Establish standard curves using known mixtures of wild-type and mutant templates
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Validate primer specificity and amplification efficiency
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Isolate mitochondria and extract RNA using RNase treatment to remove surface-bound RNA
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Next-Generation Sequencing: Provides comprehensive detection of all variants with accurate quantification of heteroplasmy levels:
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Digital droplet PCR: Offers absolute quantification without the need for standard curves:
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Partitions the PCR reaction into thousands of droplets
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Each droplet is analyzed for the presence/absence of target sequences
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Provides highly accurate heteroplasmy measurements, especially for low-level variants
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When establishing quantification methods, validation with known standards is essential. For example, mixing plasmids encoding wild-type and mutant genes at defined ratios (0-100%) can create a standard curve to validate the accuracy of your quantification method .
What is the significance of MT-ND3 mutations in mitochondrial diseases, and how can recombinant proteins be used in therapeutic approaches?
MT-ND3 mutations have significant implications in mitochondrial diseases:
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Disease associations: Mutations in MT-ND3 can cause mitochondrial complex I deficiency, which manifests as Leigh syndrome, a severe neurodegenerative disorder characterized by progressive loss of mental and movement abilities .
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Pathogenic mechanisms: Mutations disrupt complex I assembly and function, leading to:
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Decreased ATP production
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Increased reactive oxygen species (ROS) generation
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Impaired cellular respiration
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Metabolic dysfunction
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Therapeutic approaches using recombinant MT-ND3:
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mRNA therapy: Delivering wild-type MT-ND3 mRNA to mitochondria using carriers like MITO-Porter has shown promise in reducing mutation rates and improving respiratory function in patient-derived fibroblasts .
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Protein replacement: Direct delivery of recombinant MT-ND3 protein could potentially rescue complex I function.
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Gene therapy: Addressing nuclear genes that modify the penetrance or expression of MT-ND3 mutations.
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In a groundbreaking study, researchers demonstrated that mitochondrial delivery of wild-type ND3 mRNA to cells from a Leigh syndrome patient with a T10158C mutation in MT-ND3 reduced mutation rates from approximately 80% to as low as 10%, depending on the dose . This approach also improved mitochondrial respiratory function, suggesting potential therapeutic applications.
How does MT-ND3 sequence conservation vary across species, and what implications does this have for using S. hispidus as a model?
MT-ND3 sequence analysis across species reveals important evolutionary patterns:
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Conservation levels: While specific data for S. hispidus MT-ND3 is limited in the provided sources, mitochondrial genes generally show varying degrees of conservation across mammalian species. Core functional domains tend to be highly conserved.
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Species comparisons:
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Human MT-ND3 is well-characterized and serves as a reference for most studies
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Oncorhynchus kisutch (Coho salmon) MT-ND3 has been studied, with its full amino acid sequence determined (116 amino acids)
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Cotton rat (S. hispidus) mitochondrial DNA has been analyzed for population genetics, though specific MT-ND3 sequence data is not provided in the search results
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Implications for model systems:
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S. hispidus could potentially serve as a mammalian model for studying MT-ND3 function and mutations
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The utility of S. hispidus as a model depends on the conservation of key functional domains and residues compared to human MT-ND3
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Comparative studies using mtDNA analysis approaches that have been successful in S. hispidus population genetics could be adapted for MT-ND3 specific investigations
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When developing S. hispidus as a model for MT-ND3 research, researchers should perform phylogenetic analyses to determine sequence homology with human MT-ND3 and identify conserved functional domains that would make findings translatable to human disease applications.
What methods can be used to study the population genetics of MT-ND3 variants in natural Sigmodon hispidus populations?
Based on successful mtDNA studies in cotton rats, several approaches can be applied to study MT-ND3 variants:
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Restriction site variation analysis: This approach has been used effectively to reveal significant spatial and temporal heterogeneity in mtDNA genotypes among cotton rat nest sites. By focusing specifically on restriction sites affecting the MT-ND3 gene, researchers can track variants in natural populations .
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Microgeographic sampling strategies: Collecting samples from defined areas (e.g., 3.2-hectare field studies) can reveal spatial patterns in MT-ND3 variant distribution. This approach has successfully identified significant heterogeneity in mtDNA genotypes in cotton rat populations .
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Matrilineal kinship analysis: Since mtDNA is maternally transmitted, MT-ND3 variants can be used to trace matrilineal relationships and family units within populations. This offers novel perspectives on microgeographic population structure .
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Temporal sampling: Collecting samples at different time points to track changes in variant frequencies over time can reveal selection pressures or drift effects on MT-ND3 variants.
