Recombinant Human Mitochondrial 2-oxoglutarate/malate carrier protein (SLC25A11)
Description
Recombinant Human Mitochondrial 2-Oxoglutarate/Malate Carrier Protein (SLC25A11): Overview
SLC25A11, also known as the mitochondrial 2-oxoglutarate/malate carrier protein (OGCP), is a membrane-bound transporter encoded by the SLC25A11 gene. It facilitates the electroneutral exchange of 2-oxoglutarate (α-ketoglutarate) for malate, succinate, or other dicarboxylic acids across the inner mitochondrial membrane . This function is critical for metabolic processes, including the malate-aspartate shuttle (MAS), gluconeogenesis, nitrogen metabolism, and mitochondrial glutathione (GSH) transport .
Key Features
Recombinant SLC25A11 is produced in heterologous systems (e.g., wheat germ, E. coli) for research applications, including SDS-PAGE, Western blotting (WB), and ELISA .
2.1. Protein Structure
SLC25A11 belongs to the mitochondrial carrier family (TC 2.A.29), characterized by six transmembrane helices and a conserved mitochondrial carrier motif . Recombinant versions are often produced with tags (e.g., GST, His) for purification and solubility .
Expression Systems
| Host System | Tag | Purity | Applications | Reference |
|---|---|---|---|---|
| Wheat germ | None | Full-length | SDS-PAGE, ELISA, WB | |
| E. coli | GST/His | >95% | WB, IP, SDS-PAGE |
2.2. Metabolic Roles
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Malate-Aspartate Shuttle (MAS): Regenerates cytosolic NADH for mitochondrial ATP production by shuttling 2-oxoglutarate out and malate in .
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Mitochondrial GSH Transport: Facilitates GSH import into mitochondria, reducing reactive oxygen species (ROS) .
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Apoptosis Regulation: Modulates mitochondrial GSH levels; knockdown induces apoptosis in cancer cells but not normal cells .
3.1. Cancer Implications
a. Tumor Suppression and Metastasis
SLC25A11 knockdown in non-small cell lung cancer (NSCLC), melanoma, and liver cancer cells reduces ATP production, inhibits mTOR signaling, and suppresses proliferation . Key observations:
b. Genetic Mutations
Germline SLC25A11 mutations are linked to metastatic paraganglioma (pheochromocytoma) and exhibit pseudohypoxic/hypermethylator phenotypes, similar to SDHx-related tumors .
Disease Associations and Pathways
Experimental Applications and Limitations
Recombinant Protein Use
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Applications:
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Limitations:
b. In Vivo Models
SLC25A11 knockout mice exhibit embryonic lethality, while conditional knockdown in cancer cells selectively induces apoptosis without affecting normal fibroblasts .
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them in your order remarks. We will fulfill your request if possible.
Note: Our proteins are typically shipped with standard blue ice packs. If you require dry ice shipping, please communicate this in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
- Network analyses have identified SLC25A11 expression in the temporal cortex of patients with late-onset Alzheimer's disease. PMID: 28242297
- OGC serves as a model protein for understanding the transport mechanism of mitochondrial carriers. PMID: 23054077
- OGCP undergoes degradation through proteasome and lysosome degradation pathways. The degradation of parkin protein can promote the degradation of OGCP. PMID: 21500544
- Regulation of MISC-1/OGC function allows for control of mitochondrial morphology and cell survival decisions based on the metabolic needs of the cell. PMID: 21448454
- Data provide evidence for a role of the 2-oxoglutarate carrier as a glutathione transporting polypeptide PMID: 12939596
- Porphyrin accumulation in mitochondria is mediated by OGC, and porphyrins can competitively inhibit 2-oxoglutarate uptake into mitochondria. PMID: 16920706
Q&A
Basic Research Questions
What is the primary biochemical function of SLC25A11 in mitochondrial metabolism?
SLC25A11 facilitates the electroneutral exchange of 2-oxoglutarate (2-OG) and malate across the mitochondrial inner membrane, a critical step in the malate-aspartate shuttle (MAS). This shuttle regenerates mitochondrial NAD by transferring cytosolic reducing equivalents into the matrix, enabling sustained activity of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation .
Methodological Insight:
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Transport Assays: Use purified recombinant SLC25A11 reconstituted into liposomes to measure C-labeled 2-OG/malate exchange rates under varying pH and substrate concentrations .
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Genetic Knockout Models: CRISPR-Cas9-generated Slc25a11 Δ/Δ immortalized mouse chromaffin (imCC) cells exhibit disrupted MAS, leading to cytosolic NADH accumulation and pseudohypoxia .
How can researchers validate SLC25A11 expression and localization in cellular models?
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Immunohistochemistry (IHC): Use validated anti-SLC25A11 antibodies on paraffin-embedded tissues. Loss of staining correlates with biallelic inactivation (germline mutation + LOH) .
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Subcellular Fractionation: Isolate mitochondrial fractions via differential centrifugation, followed by Western blotting with compartment-specific markers (e.g., COX IV for mitochondria, GAPDH for cytosol) .
What standard techniques are used to study SLC25A11 transport kinetics?
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Isotopic Flux Measurements: Incubate mitochondria with C-labeled 2-OG and quantify metabolite exchange via LC-MS.
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Electrophysiology: Patch-clamp recordings of proteoliposomes incorporating SLC25A11 to assess voltage-dependent transport .
Advanced Research Questions
How do SLC25A11 mutations drive pseudohypoxic and hypermethylator phenotypes in tumors?
Key Findings:
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Pseudohypoxia: SLC25A11 loss disrupts MAS, causing NAD/NADH imbalance and HIF-1α stabilization even under normoxia .
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Hypermethylation: Impaired 2-OG transport reduces substrate availability for TET/JmjC-domain demethylases, leading to global DNA/histone hypermethylation (e.g., loss of 5-hmC, H3K9me3 retention) .
Methodological Approaches:
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CRISPR-Cas9 Models: Generate Slc25a11 Δ/Δ imCC cells and profile metabolites via GC-MS .
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Methylation Arrays: Compare genome-wide 5-hmC/5-mC levels in SLC25A11-mutant vs. SDHx-mutant tumors using Infinium MethylationEPIC arrays .
What contradictions exist in SLC25A11’s role in ferroptosis, and how can they be resolved?
Data Conflict:
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Study links SLC25A11 loss to tumorigenesis via pseudohypoxia, while implicates SLC25A11-FUNDC2 interactions in regulating mitochondrial glutathione (mitoGSH) and ferroptosis sensitivity.
Resolution Strategies:
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Context-Specific Analysis: Use tissue-specific knockout models (e.g., chromaffin vs. epithelial cells) to dissect metabolic rewiring.
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Integrated Omics: Perform parallel metabolomics (mitoGSH/GSSG ratios) and transcriptomics in SLC25A11-deficient cells under oxidative stress .
How can researchers address technical challenges in producing functional recombinant SLC25A11?
Challenges:
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Membrane Protein Solubility: SLC25A11 requires detergents (e.g., DDM) for extraction but may lose activity during purification.
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Functional Reconstitution: Liposome incorporation often yields <30% active protein.
Optimized Protocol:
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Expression: Use Pichia pastoris with codon-optimized SLC25A11 for high-yield mitochondrial targeting.
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Purification: Immobilized metal-affinity chromatography (IMAC) with a C-terminal His-tag.
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Activity Validation: Compare transport rates with native mitochondrial extracts .
Data Integration Tables
Table 1. Clinico-Molecular Features of SLC25A11-Mutant PPGLs
| Patient | Mutation Type | LOH | Metastasis | OGC IHC | 5-hmC Loss |
|---|---|---|---|---|---|
| 1 | c.715C>A | Yes | Yes | Negative | Yes |
| 2 | p.Ala236Ala | Yes | No | Negative | No |
| 3 | Frameshift | Yes | Yes | Negative | Yes |
| Data sourced from ; LOH = Loss of heterozygosity. |
Table 2. Comparative Phenotypes of SLC25A11 vs. SDHx Mutations
| Feature | SLC25A11 Mutants | SDHB Mutants |
|---|---|---|
| Metastasis Rate | 71% (5/7) | 40–50% |
| 5-hmC Loss | 83% (5/6) | 100% |
| HIF-1α Stabilization | Yes | Yes |
| Adapted from . |
Methodological Recommendations
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For Transport Studies: Combine isotopic tracing with computational modeling (e.g., COPASI) to simulate MAS dynamics.
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For Cancer Models: Use orthotopic xenografts of SLC25A11-KO cells to assess metastatic potential in vivo.
