SLC25A36 Antibody

What is SLC25A36 and what cellular functions does it perform?

SLC25A36 is a mitochondrial transporter that primarily imports and exports pyrimidine nucleotides into and from mitochondria. It selectively transports cytosine, guanosine, inosine, and uridine (deoxy)nucleoside mono-, di-, and triphosphates through an antiport mechanism, though it can also catalyze uniport at a much lower rate . Functionally, SLC25A36 imports deoxynucleoside triphosphates in exchange for intramitochondrial deoxynucleoside mono- and diphosphates, providing necessary precursors for de novo synthesis of mitochondrial DNA and RNA while exporting their catabolic products . This carrier plays a crucial role in mitochondrial genome maintenance, regulation of mitochondrial membrane potential, and mitochondrial respiration .

What are the available antibody options for detecting SLC25A36 in human samples?

Multiple validated antibodies are currently available for SLC25A36 detection in human samples. These include:

The rabbit polyclonal antibody ab154559 targets a recombinant fragment within human SLC25A36 from amino acid 50 to the C-terminus . The mouse monoclonal antibodies (67896-6-PBS and 67896-5-PBS) are provided in PBS only (BSA and azide free) storage buffer at 1 mg/mL, making them ready for conjugation in various applications including ELISAs, multiplex assays, mass cytometry, and multiplex imaging applications .

How does SLC25A36 differ from other mitochondrial nucleotide transporters?

SLC25A36 and SLC25A33 are the only two known mitochondrial pyrimidine nucleotide carriers in humans, sharing approximately 61% amino acid identity . While both transport pyrimidine nucleotides, they exhibit distinct substrate preferences and transport capabilities. SLC25A36 transports deoxynucleoside mono-, di-, and tri-phosphates of cytosine, uracil, inosine, and guanine, with preference for cytosine and uracil bases . In contrast, SLC25A33 transports thymine, cytosine, and uracil as deoxynucleoside di- and triphosphates, preferentially handling uridine and thymidine phosphates . Neither transporter can accommodate adenine nucleotides . Physiologically, SLC25A36 demonstrates greater activity than SLC25A33 in normal cellular function . Additionally, SLC25A36 shows tissue-specific expression patterns, with notable presence in the large intestine, brain, thyroid gland, and pancreas, but minimal expression in the liver .

What are the optimal experimental conditions for using SLC25A36 antibodies in Western blotting?

When performing Western blot analysis with SLC25A36 antibodies, researchers should consider the following methodological guidance:

• Sample preparation: Since SLC25A36 is a mitochondrial protein, enrichment of mitochondrial fractions may enhance detection sensitivity. Standard lysis buffers containing protease inhibitors are recommended to prevent degradation.

• Protein loading: Load 20-30 μg of total protein per lane, increasing to 50-60 μg when working with tissue samples with potentially lower expression levels.

• Detection considerations: Wild-type SLC25A36 has a calculated molecular mass of approximately 34.22 kDa and typically appears as a single immunoreactive band at this size when using anti-SLC25A36 polyclonal antibodies . The truncated p.Ser269llefs*35 mutant has a calculated molecular mass of 33.36 kDa but may be difficult to detect due to its rapid degradation in cells .

• Controls: Include both positive controls (cells known to express SLC25A36, such as brain-derived cell lines) and negative controls (either liver-derived cells with minimal expression or SLC25A36 knockdown cells). Additionally, use loading controls such as the β-subunit of the ATP synthase complex, which has been validated in previous studies .

• Troubleshooting: If detection proves difficult, consider longer exposure times or signal enhancement systems, as SLC25A36 expression levels may vary significantly between tissues.

How can researchers distinguish between SLC25A36 and SLC25A33 in functional studies?

Distinguishing between these closely related transporters requires careful experimental design:

• Substrate specificity analysis: Utilize transport assays with radiolabeled nucleotides to exploit the differential substrate preferences. SLC25A36 preferentially transports cytosine and uracil nucleotides, while SLC25A33 preferentially handles uridine and thymidine phosphates .

• Reconstitution in proteoliposomes: For definitive functional characterization, reconstitute purified proteins into proteoliposomes and measure substrate transport directly. This approach has been successfully used to evaluate the transport activity of wild-type and mutant SLC25A36 .

• Expression knockdown validation: When using RNA interference or CRISPR-based approaches, confirm specificity of knockdown/knockout by measuring expression levels of both SLC25A33 and SLC25A36 to rule out off-target effects.

• Physiological readouts: Monitor distinct downstream effects, such as changes in mtDNA levels, mitochondrial membrane potential, and GSH/GSSG ratios, which may be differentially affected by SLC25A36 versus SLC25A33 depletion .

• Rescue experiments: Perform cross-complementation studies in which SLC25A33 is expressed in SLC25A36-deficient cells (and vice versa) to assess functional redundancy and specificity.

What considerations should be made when using SLC25A36 antibodies for immunohistochemistry applications?

When designing immunohistochemistry experiments with SLC25A36 antibodies, researchers should consider:

• Fixation protocol: Standard formalin fixation and paraffin embedding (FFPE) appears compatible with SLC25A36 detection, as the ab154559 antibody is validated for IHC-P applications .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended to expose epitopes that may be masked during fixation.

• Tissue selection: Consider the differential expression pattern of SLC25A36 across tissues. High expression is expected in large intestine, brain, thyroid gland, and pancreas, with minimal expression in liver tissue . These expression patterns can serve as internal positive and negative controls.

• Co-staining approach: Combine SLC25A36 antibodies with mitochondrial markers (e.g., TOM20, COX IV) to confirm mitochondrial localization and with cell-type specific markers to identify expressing cell populations within heterogeneous tissues.

• Signal amplification: For tissues with lower expression levels, consider using signal amplification systems such as tyramide signal amplification to enhance detection sensitivity.

What is known about SLC25A36 deficiency in human pathology?

The first known case of SLC25A36 deficiency was reported in 2021 in a 12-year-old patient presenting with multiple clinical manifestations . The patient exhibited:

• Endocrine abnormalities: Hypothyroidism and hyperinsulinism

• Metabolic disturbances: Hyperammonemia

• Gastrointestinal issues: Chronic obstipation

• Growth parameters: Short stature

• Developmental issues: Language and general developmental delay

Whole exome sequencing identified a homozygous mutation (c.803dupT, p.Ser269llefs*35) in the SLC25A36 gene, with both parents being heterozygous carriers . Functional analysis of this mutant protein in reconstituted proteoliposomes demonstrated virtually abolished transport activity compared to wild-type SLC25A36 . Furthermore, immunoblotting revealed that the mutant protein undergoes rapid degradation in the patient's cells, as it was undetectable while being readily visible in control fibroblasts .

This case suggests SLC25A36 plays a critical role in hormonal regulation and development, potentially through its effects on mitochondrial function and nucleotide metabolism.

How might SLC25A36 antibodies be utilized in clinical research and diagnostics?

SLC25A36 antibodies offer several applications in clinical research and potential diagnostics:

• Protein expression analysis: Western blotting and immunohistochemistry with SLC25A36 antibodies can help evaluate protein expression levels in patient-derived tissues and cells, potentially identifying cases of protein deficiency or mislocalization .

• Functional validation: Following genetic identification of potentially pathogenic SLC25A36 variants, antibodies can be used to assess protein stability, as demonstrated in the case report where immunoblotting revealed degradation of the mutant protein .

• Response to therapy monitoring: In the reported case, oral uridine supplementation led to clinical improvement . SLC25A36 antibodies could potentially monitor changes in protein expression or downstream effects during therapeutic interventions.

• Research into related disorders: Given SLC25A36's role in mitochondrial function, antibodies may be valuable for investigating its potential involvement in other disorders with mitochondrial dysfunction, particularly those affecting high-expression tissues like brain, intestine, thyroid, and pancreas.

• Biomarker development: The expression pattern or post-translational modifications of SLC25A36 could potentially serve as biomarkers for mitochondrial dysfunction in specific tissues.

What experimental approaches can validate the role of SLC25A36 in mitochondrial genome maintenance?

Several methodological approaches can address this question:

• mtDNA quantification: Quantitative PCR comparing mitochondrial to nuclear DNA ratios in cells with normal versus depleted/mutated SLC25A36. Previous studies in mouse embryonic stem cells demonstrated reduced relative mtDNA amounts in SLC25A36 knockdown cells .

• Nucleotide pool analysis: Liquid chromatography-mass spectrometry (LC-MS) to measure intramitochondrial versus cytosolic nucleotide pools, particularly pyrimidine nucleotides, in the presence and absence of functional SLC25A36.

• Mitochondrial transcription assessment: Quantitative analysis of mitochondrial transcripts using RT-qPCR or RNA-seq to determine if SLC25A36 deficiency affects mitochondrial gene expression.

• In organello DNA synthesis: Isolated mitochondria can be incubated with radiolabeled nucleotides to assess de novo mtDNA synthesis rates in the presence or absence of functional SLC25A36.

• MtDNA mutation analysis: Next-generation sequencing to determine if SLC25A36 deficiency leads to increased mtDNA mutation rates due to imbalanced nucleotide pools.

How can researchers investigate the oxidative stress phenotype associated with SLC25A36 deficiency?

Previous studies in mouse embryonic stem cells demonstrated that SLC25A36 knockdown resulted in oxidative stress . Researchers can investigate this phenotype through:

• Glutathione measurement: Quantify reduced (GSH) and oxidized (GSSG) glutathione levels and calculate the GSH/GSSG ratio. In SLC25A36 knockdown mouse cells, GSH levels were decreased while GSSG levels increased, resulting in a reduced ratio indicative of oxidative stress .

• Reactive oxygen species detection: Use fluorescent probes such as DCFDA, MitoSOX, or CellROX to measure general intracellular ROS or mitochondria-specific superoxide production in cells with normal versus deficient SLC25A36.

• Protein oxidation analysis: Measure carbonyl groups or other oxidative protein modifications using approaches such as OxyBlot to assess the extent of protein damage.

• Lipid peroxidation assessment: Quantify malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) levels as markers of lipid peroxidation.

• Antioxidant enzyme activity: Measure the activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase to determine if compensatory mechanisms are activated.

• Mitochondrial function parameters: Analyze mitochondrial membrane potential using JC-1 or TMRE dyes, oxygen consumption rate with Seahorse technology, and ATP production to correlate oxidative stress with functional consequences.

What strategies can be employed to study the tissue-specific effects of SLC25A36 deficiency?

Given the differential expression of SLC25A36 across tissues and the diverse clinical manifestations observed in deficiency, tissue-specific approaches are warranted:

• Tissue-specific conditional knockout models: Generate animal models with tissue-specific Cre-loxP-mediated SLC25A36 deletion to investigate organ-specific phenotypes.

• Organoid models: Develop organoids from intestinal, thyroid, pancreatic, or neural tissues with SLC25A36 deficiency to study tissue-specific effects in controlled in vitro systems.

• Cell-type specific analysis in heterogeneous tissues: Employ single-cell RNA sequencing or fluorescence-activated cell sorting (FACS) followed by molecular analysis to identify which specific cell types within organs are most affected by SLC25A36 deficiency.

• Metabolomic profiling: Perform comparative metabolomic analysis across multiple tissues in models of SLC25A36 deficiency to identify tissue-specific metabolic derangements.

• Tissue-specific rescue approaches: Test the efficacy of uridine supplementation or other interventions in normalizing function across different affected tissues, as demonstrated by the clinical improvement in thyroid function, obstipation, growth, and development in the reported patient .

How might the interaction between SLC25A36 and SLC25A33 be investigated to understand functional redundancy?

Given that SLC25A36 and SLC25A33 are the only two known mitochondrial pyrimidine nucleotide carriers with 61% sequence identity , investigating their potential functional overlap requires:

• Double knockdown/knockout experiments: Generate cells or animal models with deficiency in both transporters to assess synergistic or additive effects compared to single deficiencies.

• Cross-complementation studies: Express SLC25A33 in SLC25A36-deficient models (and vice versa) to determine the extent of functional rescue.

• Tissue-specific expression analysis: Compare expression patterns of both transporters across tissues using quantitative approaches to identify regions of potential compensation or vulnerability.

• Structural biology approaches: Employ cryo-electron microscopy or other structural techniques to compare the substrate binding pockets and transport mechanisms of both proteins.

• Evolutionary analysis: Examine the presence and function of these transporters across species to understand their evolutionary relationship and potential subfunctionalization.

What therapeutic implications arise from understanding SLC25A36 function and pathology?

The successful treatment of the first identified SLC25A36-deficient patient with oral uridine highlights potential therapeutic approaches:

• Uridine supplementation optimization: Determine optimal dosing, delivery methods, and formulations of uridine for treating SLC25A36 deficiency.

• Alternative nucleotide precursors: Investigate whether other pyrimidine precursors or derivatives might provide therapeutic benefit.

• Antioxidant therapies: Given the observed oxidative stress in SLC25A36-deficient cells , targeted antioxidant approaches, such as vitamin C supplementation, could be beneficial.

• Gene therapy approaches: Develop viral vector-based or other gene therapy strategies to restore functional SLC25A36 in affected tissues.

• Small molecule modulator screening: Identify compounds that might enhance the residual activity of mutant SLC25A36 or upregulate compensatory pathways.

• Combination therapies: Explore synergistic approaches combining nucleotide supplementation with antioxidants or other mitochondrial support strategies.