Glutathione (GSH), the most abundant low-molecular-weight thiol in cells, plays a crucial role in life. It is a core molecule that maintains cellular redox balance, protecting cells from oxidative damage by scavenging oxidative species such as reactive oxygen species (ROS). GSH also actively participates in detoxification processes, binding to a variety of exogenous and endogenous toxic substances and promoting their excretion. Furthermore, GSH plays a key role in various physiological processes, including signal transduction, cell proliferation, and apoptosis regulation.
Given the central position of GSH in the field of life sciences, this review aims to systematically sort out the synthesis pathway of GSH, the complex metabolic network that intersects with it, and the latest clinical applications and research progress, providing a complete knowledge framework for subsequent in-depth research on the functional mechanism of GSH and the development of treatment strategies for related diseases.
How is Glutathione Synthesized? Key Precursors and Enzymes
Precursor molecules and key enzymes
The synthesis of GSH depends on three precursor molecules: glutamate, cysteine and glycine, and this process is catalyzed by key enzymes. The specific functions of each component are as follows:
| Precursor | Effect | Key enzymes |
|---|---|---|
| glutamate | Provides a γ-carboxyl group for γ-glutamylcysteine | Gamma-glutamylcysteine synthetase (GCLC, also known as GCL) |
| Cysteine | Sulfur donor, determines the rate of GSH synthesis | Same as above |
| Glycine | Provide terminal amino group to complete the formation of tripeptide GSH | Glutathione synthetase (GS) |
GCL is composed of a heavy chain (GCLC) and a light chain (GCLM). It catalyzes the first step of glutathione synthesis, which consumes one molecule of ATP and combines glutamate and cysteine to form γ-glutamylcysteine (γ-Glu-Cys). GS then catalyzes the second step, consuming another molecule of ATP to add glycine to γ-Glu-Cys, ultimately forming GSH.
Synthesis process
GSH biosynthesis follows a typical two-step, ATP-dependent enzymatic reaction. The first step is mediated by γ-glutamylcysteine synthetase (GCLC), which catalyzes the γ-amide bond between the γ-carboxyl group of glutamate and the amino group of cysteine to form γ-glutamyl-cysteine (γ-Glu-Cys). This step is generally considered the rate-limiting step, with the availability of cysteine and the expression level of GCLC jointly determining the rate of γ-Glu-Cys production.
The second stage is completed by glutathione synthetase (GS), whose substrate γ-Glu-Cys undergoes peptide bond condensation with glycine, accompanied by the hydrolysis of ATP to provide the required reaction energy. The product is reduced glutathione (GSH). This dual-enzyme cascade is widely present in the cytoplasm and is highly sensitive to oxidative stress. It can be coordinated by transcription factors (such as Nrf2) and amino acid transport systems.
Glutathione's Role in Metabolic Pathways
The metabolism of GSH does not occur in isolation; it is intertwined and closely linked with multiple core metabolic pathways to jointly maintain the normal physiological functions of cells.
Figure 1. The metabolic pathways of GSH.
Transsulfuration
The transsulfurization pathway plays a key role in supplying cysteine for GSH synthesis. The conversion of homocysteine to cysteine is accomplished by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH/CSE), providing a critical sulfur source for GSH synthesis.
At the same time, transsulfurization metabolism is closely linked to homocysteine metabolism. Homocysteine can enter the transsulfurization pathway via the CBS, which, on the one hand, reduces plasma homocysteine levels and reduces the risk of hyperhomocysteinemia-related diseases; on the other hand, it increases intracellular cysteine reserves, thereby indirectly promoting GSH synthesis and enhancing the cell's antioxidant and detoxification capabilities.
GSH‑GSSG redox cycle
The reversible conversion between glutathione (GSH) and its oxidized form, glutathione disulfide (GSSG), constitutes one of the most core cellular redox buffering systems. This cycle plays a key role in maintaining the intracellular reducing environment, protecting protein sulfhydryl groups, and regulating oxidative signaling by rapidly responding to changes in reactive oxygen species (ROS) levels.
During the oxidation phase, accumulated ROS (such as hydrogen peroxide H₂O₂ and superoxide anion O₂⁻) in the cell can directly react with GSH in a non-enzymatic manner, or, under the catalysis of glutathione peroxidase (GPX), promote the oxidative coupling of two GSH molecules to form one molecule of GSSG, while releasing two protons (H⁺) and two electrons (e⁻). This reaction not only converts toxic oxidants into harmless substances (such as water) but also creates conditions for the subsequent reduction process by storing electrons in disulfide bonds. The reaction formula can be summarized as follows:
2 GSH → GSSG + 2H⁺ + 2e⁻ (under the action of GPX or ROS)
However, to prevent GSSG accumulation from disrupting the intracellular sulfhydryl balance, cells need to rapidly convert it back to GSH through an efficient reduction system. During this reduction phase, glutathione reductase (GR) plays a central role. Using NADPH as an electron donor, it breaks the disulfide bond of GSSG and reduces it to two GSH molecules, thereby maintaining a high GSH/GSSG ratio within the cell. The reaction mechanism is as follows:
GSSG + NADPH + H⁺ → 2 GSH + NADP⁺ (catalyzed by GR)
It's worth emphasizing that the GSH/GSSG ratio is a crucial indicator of cellular redox status. In most healthy cells, this ratio is maintained in a highly reduced state between 30:1 and 100:1. A decrease in this ratio indicates increased oxidative stress, potentially triggering stress response programs such as apoptosis, necrosis, or autophagy.
The efficient operation of this cycle is highly dependent on a continuous supply of NADPH. As the primary source of reducing equivalents, NADPH is primarily produced by glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway (PPP). Furthermore, NADPH is replenished by the isocitrate dehydrogenase (IDH1/IDH2) branch of the citric acid cycle (TCA cycle), malic enzyme (ME1), and one-carbon metabolism (such as the folate cycle) depending on cell type or nutritional status. These metabolic pathways form the "energy backend" of the GSH-GSSG cycle, ensuring that GR remains catalytically active, thereby preserving the cell's redox buffering capacity.
In summary, the GSH-GSSG redox cycle does not exist in isolation, but rather operates as a dynamic equilibrium system that is highly coupled to the NADPH metabolic network. Its integrity not only determines cellular tolerance to oxidative damage but is also closely related to metabolic adaptability, drug sensitivity, and the aging process.
Detoxification pathway
GSH plays a central role in cellular detoxification, primarily through the GSH-conjugation reaction. Glutathione S-transferase (GST) catalyzes the binding of GSH to electrophilic exogenous compounds (such as carcinogens and drugs) to form soluble GSH-conjugates. These conjugates are then excreted through bile or urine, achieving detoxification.
In drug metabolism, GSH conjugation is a key "Phase II" metabolic step. It works synergistically with the cytochrome P450 system, which is responsible for metabolizing drugs and other substances through "Phase I" (e.g., oxidation and reduction reactions), imparting certain polarity and reactivity. GSH then further enhances its water solubility through "Phase II" conjugation, ultimately completing the detoxification network and promoting the excretion of drugs and other substances.
Regulatory Pathways and Transformation in GSH Metabolism
GSH metabolism also has specialized branches and is precisely regulated by multiple regulatory mechanisms to adapt to the needs of different tissues, organelles and cell states.
| Tissue/Organelle | Key Features | Major regulatory/transport molecules |
|---|---|---|
| Mitochondria | - It accounts for about 10-15% of the total cellular GSH and is an important antioxidant reservoir in the cell. | SLC25A39 is a mitochondrial inner membrane carrier responsible for transporting cytoplasmic GSH into mitochondria. |
| - Maintains mitochondrial oxidative phosphorylation, assembly of iron-sulfur proteins and normal functioning of OXPHOS. | SLC25A40 is its homologue, which is expressed at a low level but can compensate for A39 deficiency in some cells. | |
| AFG3L2-mediated protein degradation and iron-sulfur cluster feedback regulation form a GSH- dependent homeostatic mechanism. | ||
| Plant cells | - GSH is mainly distributed in the cytoplasm, chloroplasts and mitochondria, with the cytoplasm accounting for 80-85%. | The synthases GSH1 (γ-glutamylcysteine synthetase) and GSH2 (glutathione synthetase) are both single-copy genes, and their deletion is lethal. |
| - Involved in photosynthesis, metal homeostasis, antioxidant defense, and secondary metabolism. | Glutathione transferase (GST) conjugates GSH with harmful compounds, promotes cell vacuoles or exosome transport, and participates in anti-adversity defense. | |
| Plant-specific GSH derivatives (such as allicin S-allyl -cysteine sulfoxide) play a role in drought resistance, salt tolerance, and disease and pest resistance. | ||
| Endoplasmic reticulum (ER) | - It accounts for about 5-10% of the total cellular GSH and provides a reducing environment for protein folding and disulfide bond formation. | - GSH and GSSG are exchanged across the membrane through transporters such as gamma -glutamyl transpeptidase (GGT) and SLC35A2. |
| Cell nucleus | - GSH is enriched in the nucleus and regulates DNA repair, cell cycle and proliferation. | - Mainly relies on passive diffusion and local transport proteins in the nuclear pore complex, and the specific mechanism is still under investigation. |
| Chloroplasts | - Provides reducing power for photosynthesis and participates in the protection of photosystem I/II. | Cytoplasmic GSH is transported into chloroplasts via chloroplast membrane carriers (not yet fully identified), partly dependent on the regulation of GSH S-transferase. |
Regulatory factors and signaling networks
The Nrf2-ARE axis is a key signaling pathway regulating GSH synthesis and metabolism. Under oxidative stress, the transcription factor Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) translocates from the cytoplasm into the nucleus and binds to the antioxidant response element (ARE). This binding transcriptionally upregulates the expression of a series of key genes, including GCLC, GCLM (the light chain of GCLC), and xCT (system xc⁻, responsible for cysteine uptake), thereby increasing GSH synthesis and cysteine uptake, thereby strengthening the cell's antioxidant defenses.
In addition, Nrf2 can also regulate the expression of downstream enzymes such as GST and GR, further improving the cell's antioxidant defense network and enabling cells to more effectively respond to damage caused by oxidative stress.
Glutathione's Impact on Diseases: From Sepsis to Autism
Abnormal GSH metabolism is closely related to the occurrence and development of various diseases, and also provides new targets and directions for the treatment and research of diseases.
The occurrence and development of different diseases are associated with abnormal GSH metabolism through different mechanisms:
Glutathione dynamics during sepsis
Sepsis is characterized by severe redox imbalance, and glutathione plays a key role in combating oxidative and nitrosative stress. During the acute phase of sepsis, glutathione synthesis is enhanced in the liver and other tissues, a process regulated by multiple factors, including a decreased reduced/oxidized glutathione ratio, reactive oxygen and nitrogen species, proinflammatory cytokines, heat shock proteins, and physical inactivity. However, in chronic critical illness, glutathione synthesis may be impaired by cysteine depletion, protein-energy malnutrition, hyperglycemia, pharmacological doses of glucocorticoids, and decreased anterior pituitary hormone secretion. These findings suggest that differentiated glutathione metabolism intervention strategies should be adopted for patients at different stages of sepsis.
Autism spectrum disorder
Numerous studies have shown that children with autism spectrum disorder (ASD) exhibit abnormalities in oxidative stress markers. A meta-analysis of 87 studies (9,109 participants) found that ASD children had significantly elevated blood levels of oxidative markers such as GSSG, malondialdehyde, and homocysteine, while significantly decreased levels of GSH, total glutathione, the GSH/GSSG ratio, and antioxidant substances such as cysteine, vitamin B9, and vitamin D. Alterations in glutathione metabolism biomarkers were particularly consistent and may provide clues for the early diagnosis of ASD.
Heavy metal detoxification
Glutathione and its derivatives play a central role in heavy metal detoxification. In mammals, yeast cadmium factor (YCF1) encodes a MgATP-fueled glutathione conjugate transporter responsible for transporting the di(glutathione)-cadmium complex to the vacuole for sequestration, a crucial pathway for cadmium detoxification.
Drug metabolism and toxicity
Glutathione plays a dual role in drug metabolism and detoxification. On the one hand, GSH binds to reactive metabolites, preventing them from interacting with cellular macromolecules. The classic mechanism of acetaminophen hepatotoxicity is that its reactive metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), depletes GSH and then covalently binds to proteins. On the other hand, glutathione conjugates of certain substances may be further metabolized into more toxic products. For example, the glutathione conjugate of hexachlorobutadiene is processed in the kidneys into reactive intermediates, leading to nephrotoxicity.
Advances in Glutathione Research for Disease Research
Targeted therapy for GSH metabolism has become a research hotspot, and metabolomics and proteomics technologies have also provided powerful tools for GSH-related research:
Targeting GSH metabolism: Inhibiting key molecules such as GCLC, xCT (cysteine uptake carrier) or SLC25A39 (mitochondrial GSH transporter), or using GSH-depleting nanozymes, has shown the potential to enhance ROS-mediated tumor cell death and improve related disease symptoms in preclinical models (such as cell models and animal models), providing new strategies for disease treatment.
Metabolomics/Proteomics: High-throughput mass spectrometry technology can comprehensively analyze metabolites in cells, while metabolic flux analysis can track the dynamic changes of metabolites. These technologies have been used to depict the GSH metabolic network, helping researchers to screen new drug targets (such as targets for key enzymes in GSH synthesis or metabolism) and biomarkers (such as GSH-related metabolite markers for disease diagnosis and prognosis assessment).
Conclusion
GSH is synthesized from three precursor molecules, glutamate, cysteine, and glycine, through a two-step enzymatic reaction of GCLC and GS. Its metabolism is closely linked to the transsulfurization branch (providing cysteine), the GSH-GSSG redox cycle (maintaining redox balance), and the detoxification system (completing substance detoxification), forming a complex and efficient metabolic network. Mitochondrial GSH transport (via SLC25A39) and the Nrf2-ARE regulatory pathway constitute key regulatory mechanisms at the tissue/cellular level, ensuring that GSH metabolism adapts to different physiological and pathological states.
In the future, we will develop small molecule inhibitors or activators for specific transporters such as SLC25A39 and xCT, and use gene editing tools (such as CRISPR/Cas9) to precisely edit related genes to achieve precise regulation of GSH metabolism, providing more targeted means for disease treatment.
By combining multi-omics technologies such as metabolomics, single-cell transcriptomics and spatial omics, we systematically depict the dynamic changes of GSH in different tissues (such as liver, brain, tumor tissue, etc.) and under different disease states (such as normal, inflammatory, cancerous, etc.), and deeply analyze the functional mechanism of GSH in different physiological and pathological backgrounds, providing a more comprehensive theoretical basis for the diagnosis, treatment and prognosis of diseases.
References
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- Carmel-Harel, O., & Storz, G. (2000). Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and saccharomyces cerevisiae responses to oxidative stress. Annual review of microbiology, 54, 439–461.
- Gasmi, A., Nasreen, A., et al. (2024). An Update on Glutathione's Biosynthesis, Metabolism, Functions, and Medicinal Purposes. Current medicinal chemistry, 31(29), 4579–4601.
