GO:0004791 thioredoxin-disulfide reductase (NADPH) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004791 describes the enzymatic activity that reduces oxidized thioredoxin using NADPH as the electron donor, regenerating reduced thioredoxin for cellular antioxidant defense and redox signaling.
• Thioredoxin reductase (TrxR) enzymes are selenoproteins in mammals and are essential for maintaining thioredoxin in its reduced state, thereby controlling protein thiol redox balance.
• The thioredoxin system, including thioredoxin reductase, regulates diverse processes such as DNA synthesis, apoptosis, and inflammation through thiol-disulfide exchange.
• Dysregulation of thioredoxin reductase is implicated in cancer, neurodegenerative diseases, and cardiovascular disorders, making it a pharmacological target.
• Plant and algal NADPH-thioredoxin reductases (NTRC) are regulated by calcium-binding proteins and CBSX proteins, linking redox control to metabolic and stress responses.
• CRISPR-based knockout, knock-in, and point-mutation models are powerful tools to dissect the specific roles of thioredoxin reductase genes in health and disease.
Description
Thioredoxin-disulfide reductase (NADPH) activity, classified under GO:0004791, is a fundamental molecular function that maintains cellular redox homeostasis by reducing oxidized thioredoxin. This activity is catalyzed by thioredoxin reductase (TrxR) enzymes, which transfer electrons from NADPH to the active-site disulfide of thioredoxin, regenerating the reduced dithiol form that participates in numerous redox-dependent processes. The thioredoxin system, composed of thioredoxin, thioredoxin reductase, and NADPH, is conserved across all kingdoms of life and plays critical roles in antioxidant defense, DNA synthesis, and signal transduction. In mammals, thioredoxin reductases are selenoproteins, and their activity is essential for embryonic development and cellular survival. Given its central role in redox regulation, thioredoxin-disulfide reductase activity is a focal point for researchers studying cancer, inflammation, and metabolic disorders. Understanding the molecular mechanisms, regulation, and disease associations of this activity is crucial for developing targeted therapeutic strategies and for interpreting redox-related experimental data.
thioredoxin-disulfide reductase (NADPH) activity At A Glance
| GO ID | GO:0004791 |
|---|---|
| GO term | thioredoxin-disulfide reductase (NADPH) activity |
| Ontology | molecular_function |
| Synonym | NADPH:oxidized thioredoxin oxidoreductase activity; thioredoxin disulfide reductase activity; thioredoxin-disulfide reductase activity; thioredoxin-disulfide reductase (NADP) activity; thioredoxin-disulphide reductase activity; thioredoxin reductase (NADPH) activity |
| Major function | Reduction of oxidized thioredoxin using NADPH as electron donor, maintaining cellular redox homeostasis |
| Reaction | thioredoxin-dithiol + NADP+ = thioredoxin-disulfide + H+ + NADPH |
| Cofactor | FAD (flavin adenine dinucleotide) in mammalian thioredoxin reductases |
| Localization | Cytoplasm, mitochondria, and chloroplasts (in plants) |
| EC number | 1.8.1.9 |
What Is GO:0004791?
Thioredoxin-disulfide reductase (NADPH) activity (GO:0004791) is defined as the catalysis of the reaction: thioredoxin-dithiol + NADP+ = thioredoxin-disulfide + H+ + NADPH. In other words, it is the enzyme activity that uses NADPH to reduce the disulfide bond in oxidized thioredoxin, producing reduced thioredoxin and NADP+. This activity is synonymous with NADPH:oxidized thioredoxin oxidoreductase activity, thioredoxin disulfide reductase activity, and thioredoxin reductase (NADPH) activity, among others. It is a molecular function that enables cells to maintain a reducing environment and to regulate protein function through thiol-disulfide exchange.
Why Is thioredoxin-disulfide reductase (NADPH) activity Important in Cell Biology?
Thioredoxin-disulfide reductase (NADPH) activity is essential for life because it sustains the reduced state of thioredoxin, a major cellular antioxidant and redox regulator. By controlling the redox state of thioredoxin, this activity influences a wide array of biological processes, including DNA synthesis, apoptosis, and immune responses. In mammals, thioredoxin reductase is a selenoprotein, and its dysfunction has been linked to cancer, cardiovascular diseases, and neurodegeneration. Moreover, the thioredoxin system is a target for drug development, as inhibiting thioredoxin reductase can selectively kill cancer cells that rely on high antioxidant capacity. In plants and algae, NADPH-thioredoxin reductase C (NTRC) is crucial for chloroplast redox regulation and stress tolerance. Therefore, understanding this activity is fundamental for both basic biology and translational research.
• Maintains reduced thioredoxin pools, protecting cells from oxidative stress.
• Regulates protein function through thiol-disulfide exchange, affecting enzyme activity and transcription factor binding.
• Supports DNA synthesis by providing reducing equivalents for ribonucleotide reductase.
• Modulates apoptosis and cell survival pathways, with implications for cancer therapy.
• Controls inflammatory signaling by regulating tissue factor activity.
• Influences cardiovascular health through redox-dependent mechanisms.
• Plays a role in plant and algal stress responses via chloroplast NTRC.
• Serves as a pharmacological target for cancer and other diseases.
• Is essential for embryonic development in mammals.
• Provides a model system for studying redox regulation across species.
What Happens During thioredoxin-disulfide reductase (NADPH) activity?
Electron Transfer from NADPH to Thioredoxin Reductase
In simple terms: NADPH gives electrons to thioredoxin reductase, which then passes them to thioredoxin.
The catalytic cycle begins when NADPH binds to thioredoxin reductase and transfers a hydride ion to the enzyme's FAD cofactor, reducing it to FADH2. This reduced FAD then reduces the enzyme's active-site disulfide, forming a dithiol. In mammalian thioredoxin reductases, the active site contains a selenocysteine residue that is essential for efficient catalysis. The reduced enzyme then interacts with oxidized thioredoxin, transferring electrons to reduce its disulfide bond and releasing reduced thioredoxin.
Reduction of Oxidized Thioredoxin
In simple terms: The enzyme reduces the disulfide bond in thioredoxin, turning it back into its active, reduced form.
Oxidized thioredoxin, which contains a disulfide bond in its active site (Cys-Gly-Pro-Cys), binds to reduced thioredoxin reductase. Through a series of thiol-disulfide exchange reactions, the disulfide bond in thioredoxin is reduced to a dithiol, while the enzyme's active site is oxidized. This regenerated reduced thioredoxin can then participate in various cellular processes, such as reducing oxidized proteins, donating electrons to ribonucleotide reductase, or regulating transcription factor activity.
Regeneration of Reduced Thioredoxin and Cellular Redox Homeostasis
In simple terms: By constantly reducing thioredoxin, this activity keeps the cell's internal environment reducing and protects it from damage.
The continuous cycle of thioredoxin reduction by thioredoxin reductase maintains a high ratio of reduced to oxidized thioredoxin, which is critical for cellular redox homeostasis. Reduced thioredoxin acts as a general protein disulfide reductase, reducing oxidized cysteine residues in various target proteins, thereby modulating their activity. This system also works in concert with other antioxidant systems, such as glutathione and peroxiredoxins, to defend against oxidative stress. In plants, NADPH-thioredoxin reductase C (NTRC) performs a similar function in chloroplasts, regulating enzymes involved in carbon fixation and stress responses.
Structural and Regulatory Features of Thioredoxin Reductase
In simple terms: The enzyme has a special structure that allows it to interact with NADPH and thioredoxin, and its activity can be turned on or off by other proteins.
Mammalian thioredoxin reductases are homodimeric flavoproteins, with each subunit containing an FAD-binding domain, an NADPH-binding domain, and an interface domain that mediates dimerization. The C-terminal active site contains a selenocysteine residue, which is crucial for catalysis and is sensitive to oxidation. In plants and algae, NTRC is a single polypeptide with both thioredoxin and thioredoxin reductase domains, and its activity can be regulated by calcium-binding proteins like calredoxin and CBSX proteins. These regulatory interactions allow the cell to fine-tune thioredoxin reductase activity in response to metabolic and environmental cues.
Key Genes Involved in GO:0004791 thioredoxin-disulfide reductase (NADPH) activity
The following genes encode proteins that either possess thioredoxin-disulfide reductase (NADPH) activity or directly regulate it, and they are commonly studied in redox biology.
| Gene | Major Role | Research Relevance |
|---|---|---|
| TXNRD1 | Cytosolic thioredoxin reductase 1; reduces thioredoxin-1 | Cancer, oxidative stress, drug resistance |
| TXNRD2 | Mitochondrial thioredoxin reductase 2; reduces thioredoxin-2 | Mitochondrial function, cardiovascular disease |
| TXNRD3 | Testis-specific thioredoxin reductase 3 | Spermatogenesis, redox regulation |
| TXN | Thioredoxin; substrate of thioredoxin reductase | Cell growth, apoptosis, inflammation |
| TXN2 | Mitochondrial thioredoxin; substrate of TXNRD2 | Mitochondrial redox control |
| NTRC | NADPH-thioredoxin reductase C (plants/algae); reduces chloroplast thioredoxins | Photosynthesis, stress response |
| CBSX1 | Cystathionine-β-synthase domain protein; negative regulator of NTRC | Regulation of NTRC activity |
| CBSX2 | Cystathionine-β-synthase domain protein; negative regulator of NTRC | Regulation of NTRC activity |
| CALREDOXIN | Calcium-binding protein; regulates NTRC in Chlamydomonas | Calcium signaling in chloroplast |
| PDI | Protein disulfide-isomerase; can be reduced by thioredoxin reductase | Protein folding, redox regulation |
| PRDX1 | Peroxiredoxin 1; uses thioredoxin as electron donor | Antioxidant defense |
| PRDX2 | Peroxiredoxin 2; uses thioredoxin as electron donor | Antioxidant defense |
| RRM1 | Ribonucleotide reductase subunit; requires thioredoxin for reduction | DNA synthesis |
| ASK1 | Apoptosis signal-regulating kinase 1; regulated by thioredoxin | Apoptosis, stress signaling |
| PTEN | Phosphatase and tensin homolog; redox-regulated by thioredoxin | Tumor suppression |
| NFKB1 | NF-κB subunit; regulated by thioredoxin | Inflammation, immune response |
| TF | Tissue factor; activity controlled by thioredoxin system | Coagulation, inflammation |
How Is thioredoxin-disulfide reductase (NADPH) activity Regulated?
Thioredoxin-disulfide reductase (NADPH) activity is regulated at multiple levels. In mammalian cells, TXNRD1 expression is induced by oxidative stress through the Nrf2/ARE pathway, and its activity can be inhibited by nitric oxide and other electrophiles. In plants and algae, NTRC activity is modulated by calcium-binding proteins such as calredoxin and CBSX proteins, which respond to changes in calcium and redox status. Additionally, the thioredoxin system is regulated by thioredoxin-interacting protein (TXNIP), which binds to and inhibits thioredoxin, thereby indirectly affecting thioredoxin reductase activity. Post-translational modifications, including oxidation of the selenocysteine residue, can also reversibly inactivate thioredoxin reductase.
thioredoxin-disulfide reductase (NADPH) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| TXNRD1 | Cancer, drug resistance | Knockout in cancer cell lines; overexpression for resistance studies |
| TXNRD2 | Cardiomyopathy, mitochondrial dysfunction | Cardiomyocyte-specific knockout mice |
| TXN | Inflammation, cancer | Knockout mice; point mutation of active-site cysteines |
| NTRC | Plant stress response | Arabidopsis ntrc mutants; knock-in of mutant variants |
| CBSX1 | Regulation of NTRC in plants | Knockout and overexpression in Arabidopsis |
Thioredoxin Reductase in Cancer
Thioredoxin reductase is often overexpressed in many cancers, where it supports tumor growth by maintaining redox balance and promoting cell survival. High levels of TXNRD1 are associated with resistance to chemotherapy and poor prognosis in several cancer types. Inhibitors of thioredoxin reductase, such as gold compounds and motexafin gadolinium, are being explored as anticancer agents. The thioredoxin system also regulates apoptosis through ASK1 and PTEN, linking redox control to tumor suppression.
Thioredoxin Reductase in Cardiovascular and Inflammatory Diseases
Thioredoxin and thioredoxin reductase control tissue factor activity, which is critical for coagulation and inflammation. Dysregulation of this system contributes to thrombosis and atherosclerosis. In addition, thioredoxin reductase protects against oxidative stress in the heart, and its deficiency has been linked to cardiomyopathy in animal models.
Thioredoxin Reductase in Neurodegeneration
Oxidative stress is a hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's. Thioredoxin reductase helps maintain neuronal redox balance, and its dysfunction may contribute to neuronal death. In plants and algae, NTRC deficiency leads to impaired photosynthesis and increased sensitivity to oxidative stress, providing insights into redox-related stress responses.
From thioredoxin-disulfide reductase (NADPH) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of TXNRD1 loss on cancer cell survival? | TXNRD1 knockout cancer cell lines (e.g., A549, HeLa) |
| How does mitochondrial thioredoxin reductase contribute to cardiac function? | TXNRD2 conditional knockout mice |
| What is the role of the selenocysteine residue in catalysis? | Point mutation of TXNRD1 selenocysteine to cysteine |
| How does NTRC regulate chloroplast redox? | Arabidopsis ntrc knockout and complemented lines |
| Can thioredoxin reductase be targeted for cancer therapy? | Xenograft models with TXNRD1 overexpression or knockout |
| What are the interactors of thioredoxin reductase? | Knock-in of tagged TXNRD1 for proteomics |
How to Study the thioredoxin-disulfide reductase (NADPH) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| NADPH oxidation assay | Thioredoxin reductase activity | Enzyme kinetics, inhibitor screening |
| Insulin reduction assay | Thioredoxin reductase activity | Activity in cell lysates |
| CRISPR knockout | Loss of gene function | Studying cellular phenotypes |
| roGFP imaging | Cellular redox state | Live-cell redox dynamics |
| Redox proteomics | Thioredoxin target proteins | Identifying redox-regulated pathways |
| X-ray crystallography | Three-dimensional structure | Understanding catalytic mechanism |
| RNA-seq | Transcriptional changes | Response to thioredoxin reductase inhibition |
Enzymatic Activity Assays
Thioredoxin-disulfide reductase activity is typically measured using a coupled assay with NADPH and oxidized thioredoxin, monitoring the decrease in NADPH absorbance at 340 nm. Alternatively, insulin reduction assays can be used, where reduced thioredoxin reduces insulin disulfides, leading to precipitation. These assays are essential for characterizing enzyme kinetics and inhibitor effects.
Genetic Knockout and Knockdown
CRISPR-Cas9 knockout of TXNRD1 or TXNRD2 in cell lines allows researchers to study the consequences of loss of thioredoxin reductase activity on cellular redox, proliferation, and stress responses. siRNA or shRNA knockdown provides a reversible alternative. In plants, T-DNA insertion mutants of NTRC are used to investigate its role in photosynthesis.
Redox Proteomics and Imaging
Redox-sensitive GFP (roGFP) sensors can monitor the redox state of thioredoxin in live cells. Proteomic approaches, such as thioredoxin affinity chromatography or redox Western blotting, identify target proteins whose thiols are reduced by the thioredoxin system. These methods reveal the downstream effects of thioredoxin reductase activity.
Structural and Biophysical Studies
X-ray crystallography and cryo-EM have provided insights into the structure of thioredoxin reductases, including the selenocysteine-containing active site. Isothermal titration calorimetry and surface plasmon resonance can measure binding affinities between thioredoxin reductase and its substrates or inhibitors.
How CRISPR Can Be Used to Study GO:0004791 thioredoxin-disulfide reductase (NADPH) activity
Knockout
CRISPR-Cas9 knockout of TXNRD1 or TXNRD2 generates cell lines completely lacking thioredoxin reductase activity, enabling studies of its essentiality and role in oxidative stress responses. Knockout models have revealed that TXNRD1 is required for cell survival under oxidative stress and that loss of TXNRD2 impairs mitochondrial function.
Point Mutation
Point mutations can be introduced into the active site of thioredoxin reductase, such as substituting the selenocysteine with cysteine, to dissect its catalytic mechanism and sensitivity to inhibitors. These models help distinguish between the roles of different active-site residues.
Knock-in
Knock-in of tagged versions of TXNRD1 (e.g., HA or GFP) allows for localization and interaction studies using immunofluorescence or immunoprecipitation. Knock-in of disease-associated mutations can model human disorders in cell lines or mice.
Overexpression
Overexpression of TXNRD1 or NTRC via CRISPR activation or lentiviral vectors can be used to study the effects of increased thioredoxin reductase activity on cell proliferation, drug resistance, and redox balance. Overexpression models are valuable for testing inhibitors and for understanding the contribution of thioredoxin reductase to cancer progression.
How EDITGENE Supports thioredoxin-disulfide reductase (NADPH) activity Research
Researchers studying thioredoxin-disulfide reductase (NADPH) activity-related genes often need to determine whether a candidate gene is causally involved in a specific redox-dependent process or disease. This requires precise genetic manipulation, which can be achieved through CRISPR-based knockout, point mutation, knock-in, or overexpression models. EDITGENE provides a comprehensive suite of services to generate such models, enabling rigorous functional studies of thioredoxin reductase and its regulators.
Contact EDITGENE today to design your custom CRISPR model for thioredoxin-disulfide reductase (NADPH) activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| TXNRD2 Knockout HEK293 Cell Line | EDJ-KQ7098 | Human | 10587 | Details Get a Quote |
| TXNRD3 Knockout HEK293 Cell Line | EDJ-KQ7440 | Human | 114112 | Details Get a Quote |
| SELENOT Knockout HEK293 Cell Line | EDJ-KQ11201 | Human | 51714 | Details Get a Quote |
| NXN Knockout HEK293 Cell Line | EDJ-KQ14533 | Human | 64359 | Details Get a Quote |
| TXNRD2 Knockout A-549 Cell Line | EDJ-KQ31949 | Human | 10587 | Details Get a Quote |
| TXNRD2 Knockout HeLa Cell Line | EDJ-KQ31951 | Human | 10587 | Details Get a Quote |
| NXN Knockout A-549 Cell Line | EDJ-KQ44813 | Human | 64359 | Details Get a Quote |
| NXN Knockout HCT 116 Cell Line | EDJ-KQ44814 | Human | 64359 | Details Get a Quote |
| NXN Knockout HeLa Cell Line | EDJ-KQ44815 | Human | 64359 | Details Get a Quote |
| TXNRD2 Knockout HCT 116 Cell Line | EDJ-KQ30567 | Human | 10587 | Details Get a Quote |
| TXNRD3 Knockout HeLa Cell Line | EDJ-KQ31283 | Human | 114112 | Details Get a Quote |
| TXNRD3 Knockout A-549 Cell Line | EDJ-KQ32646 | Human | 114112 | Details Get a Quote |
| TXNRD3 Knockout HCT 116 Cell Line | EDJ-KQ32647 | Human | 114112 | Details Get a Quote |
| SELENOT Knockout A-549 Cell Line | EDJ-KQ39264 | Human | 51714 | Details Get a Quote |
| SELENOT Knockout HCT 116 Cell Line | EDJ-KQ39265 | Human | 51714 | Details Get a Quote |
Frequently Asked Questions About thioredoxin-disulfide reductase (NADPH) activity
What is thioredoxin-disulfide reductase (NADPH) activity?
It is the enzymatic activity (GO:0004791) that reduces oxidized thioredoxin using NADPH, maintaining cellular redox balance.
What genes are involved in thioredoxin-disulfide reductase (NADPH) activity?
Key genes include TXNRD1, TXNRD2, TXNRD3 in mammals, and NTRC in plants and algae.
What is the reaction catalyzed by thioredoxin-disulfide reductase?
The reaction is: thioredoxin-dithiol + NADP+ = thioredoxin-disulfide + H+ + NADPH.
Why is thioredoxin reductase important in cancer?
Thioredoxin reductase is often overexpressed in cancer, supporting tumor growth and drug resistance, making it a therapeutic target.
How is thioredoxin reductase activity measured?
Common methods include NADPH oxidation assays and insulin reduction assays.
What are the synonyms for thioredoxin-disulfide reductase (NADPH) activity?
Synonyms include NADPH:oxidized thioredoxin oxidoreductase activity, thioredoxin disulfide reductase activity, and thioredoxin reductase (NADPH) activity.
How does thioredoxin reductase regulate inflammation?
It controls tissue factor activity through thiol redox-dependent mechanisms, influencing coagulation and inflammation.
What is the role of NTRC in plants?
NTRC is a chloroplast thioredoxin reductase that regulates photosynthesis and stress responses, and its activity is modulated by CBSX proteins and calredoxin.
Can thioredoxin reductase be targeted for drug development?
Yes, inhibitors of thioredoxin reductase are being explored as anticancer agents.
What CRISPR models are available for studying thioredoxin reductase?
Knockout, point mutation, knock-in, and overexpression models can be generated for TXNRD1, TXNRD2, and other related genes.
Conclusion
Thioredoxin-disulfide reductase (NADPH) activity (GO:0004791) is a central molecular function that maintains cellular redox homeostasis by reducing thioredoxin. Its importance spans antioxidant defense, DNA synthesis, apoptosis, and inflammation, with strong links to cancer, cardiovascular disease, and neurodegeneration. The thioredoxin system is conserved from mammals to plants, where NTRC plays specialized roles in chloroplast function. CRISPR-based genetic models are invaluable for dissecting the precise roles of thioredoxin reductase genes and for developing targeted therapies. EDITGENE provides comprehensive services to accelerate this research.
References
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- 3. Tran CM et al.. 2023. Cystathionine-β-synthase X proteins negatively regulate NADPH-thioredoxin reductase C activity.. Biochem Biophys Res Commun 653:47-52 PMID: 36857899
- 4. Wang P et al.. 2013. Thioredoxin and thioredoxin reductase control tissue factor activity by thiol redox-dependent mechanism.. J Biol Chem 288(5):3346-58 PMID: 23223577
- 5. Bjørklund G et al.. 2021. Thioredoxin reductase as a pharmacological target.. Pharmacol Res 174:105854 PMID: 34455077
- 6. Deschoenmaeker F et al.. 2021. Thioredoxin pathway in Anabaena sp. PCC 7120: activity of NADPH-thioredoxin reductase C.. J Biochem 169(6):709-719 PMID: 33537746
- 7. Lundström J et al.. 1990. Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity.. J Biol Chem 265(16):9114-20 PMID: 2188973
- 8. Zinzius K et al.. 2023. Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii.. Plant Physiol 193(3):2122-2140 PMID: 37474113
