Glutathione: A Samsonian life-sustaining small molecule that protects against oxidative stress, ageing and damaging inflammation

Abstract

Many local and systemic diseases especially diseases that are leading causes of death globally like chronic obstructive pulmonary disease, atherosclerosis with ischemic heart disease and stroke, cancer and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 19 (COVID-19), involve both, (1) oxidative stress with excessive production of reactive oxygen species (ROS) that lower glutathione (GSH) levels, and (2) inflammation. The GSH tripeptide (γ- L-glutamyl-L-cysteinyl-glycine), the most abundant water-soluble non-protein thiol in the cell (1-10 mM) is fundamental for life by (a) sustaining the adequate redox cell signaling needed to maintain physiologic levels of oxidative stress fundamental to control life processes, and (b) limiting excessive oxidative stress that causes cell and tissue damage. GSH activity is facilitated by activation of the Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) redox regulator pathway, releasing Nrf2 that regulates expression of genes controlling antioxidant, inflammatory and immune system responses. GSH exists in the thiol-reduced (>98% of total GSH) and disulfide-oxidized (GSSG) forms, and the concentrations of GSH and GSSG and their molar ratio are indicators of the functionality of the cell. GSH depletion may play a central role in inflammatory diseases and COVID-19 pathophysiology, host immune response and disease severity and mortality. Therapies enhancing GSH could become a cornerstone to reduce severity and fatal outcomes of inflammatory diseases and COVID-19 and increasing GSH levels may prevent and subdue these diseases. The life value of GSH makes for a paramount research field in biology and medicine and may be key against systemic inflammation and SARS-CoV-2 infection and COVID-19 disease. In this review, we emphasize on (1) GSH depletion as a fundamental risk factor for diseases like chronic obstructive pulmonary disease and atherosclerosis (ischemic heart disease and stroke), (2) importance of oxidative stress and antioxidants in SARS-CoV-2 infection and COVID-19 disease, (3) significance of GSH to counteract persistent damaging inflammation, inflammaging and early (premature) inflammaging associated with cell and tissue damage caused by excessive oxidative stress and lack of adequate antioxidant defenses in younger individuals, and (4) new therapies that include antioxidant defenses restoration.

Keywords: COVID-19; atherosclerosis; chronic obstructive pulmonary disease; glutathione; inflammaging; nuclear factor erythroid 2-related factor 2; oxidative stress; reactive oxygen species.

FIGURE 1

Glutathione (GSH) synthesis, chemical structure and different forms of GSH. (A) GSH is synthesized in the cytosol in two steps. The first step is the formation of γ-glutamylcysteine from glutamate and cysteine by the enzyme γ-glutamylcysteine synthetase (glutamate cysteine ligase). The second step in GSH synthesis is regulated by glutathione synthetase. Glutathione cysteine ligase and cysteine (green) are the limiting factors in GSH synthesis. The γ-carboxyl linkage (gray) and the sulfhydryl group (green) provide stability and reductive power to the molecule, respectively. (B) Chemical structure of reduced (GSH), oxidized (GSSG) glutathione and GS-protein generated by protein glutathionylation. Glutathione peroxidase oxidizes GSH and glutathione reductase reduces GSSG, while glutathione-S-transferase participates in protein glutathionylation.

FIGURE 2

Glutathione distribution in subcellular compartments. GSH (γ- L-glutamyl-L-cysteinyl-glycine), a water-soluble tripeptide formed by the amino-acids glutamic acid, cysteine and glycine, is considered the major non-protein low molecular weight modulator of redox processes and the most important thiol reducing agent of the cell. (1) ATP-dependent GSH biosynthesis occurs in the cytosol of the cell and cysteine (red) and glutamate cysteine ligase (gray) are rate-limiting factors for its production. (2) Extracellular GSH is enzymatically degraded on the surface of the cells by γ-glutamyl transpeptidase generating the γ-glutamyl fraction (taken into the cell as γ-glutamyl-amino acid that can be metabolized to release the amino acid and 5-oxoproline, which can then be converted into glutamate to be used in the synthesis of GSH) and the cysteinyl-glycine fraction; and by dipeptidases splitting cysteinyl-glycine generating cysteine and glycine that are taken into the cell. (3) To allow normal cell function, it is essential to maintain an optimal GSH: GSSG ratio throughout all cell compartments. (4) The inner mitochondrial membrane system transport, that involves dicarboxylate and 2-oxoglutarate anion transporters, allows the passage of negatively charged GSH from the cytosol to the mitochondria. (5) GSH is present in both reduced (GSH) and oxidized (GSSG) states, and reduced GSH is maintained by GSH reductase, a cytosolic NADPH-dependent enzyme. GSSG returns to the reduced state by the NADPH-dependent activity of glutathione reductase. NADPH is rapidly regenerated from NADP + using electrons derived from catabolism of substrate molecules, such as glucose or isocitric and malic acid (pentose phosphate pathway). Reduced GSH neutralizes cellular hydroperoxides through GSH peroxidase activity.

FIGURE 3

Glutathione synthesis: A two-step pathway. Homeostasis of cellular glutathione. Synthesis and regulation of the cell concentrations. Glutamate cysteine ligase (γ-glutamyl cysteine synthetase) constitute the first step in the synthesis of glutathione (GSH) forming γ-L-glutamyl-L-cysteine using adenosine triphosphate (ATP). Glutathione synthetase constitute the second step forming GSH, also using ATP. Cellular GSH concentration regulates the function of glutamate cysteine ligase.

FIGURE 4

The “Glutathione Pathway.” Glutathione synthesis, γ-glutamyl pathway, cellular distribution, antioxidant properties, catabolism of xenobiotics, and glutathione recycling in the cell. The figure shows a schematic representation of the “glutathione pathway.” Glutathione (GSH) is synthesized from glutamate, cysteine, and glycine by γ-glutamyl-cysteine synthetase (glutamate cysteine ligase) and glutathione synthetase. Glutathione redox state is regulated, in part, by glutathione peroxidases, forming oxidized glutathione (GSSG), and by a reaction catalyzed by glutathione reductase. Glutathione is conjugated to substrates both through the action of the glutathione S-transferases and through non-enzymatic reactions. Glutathione conjugates can be excreted from the cells by members of the ATP-binding cassette (ABC) transporter family.

FIGURE 5

Cellular glutathione synthesis and recycling: The importance of the γ-glutamyl pathway. The degradation or catabolic part of the GSH cycle, takes place partially extracellularly and partially inside cells. (1) The extracellular degradation of GSH occurs on the surface of the cells that express the enzyme γ-glutamyl transpeptidase (GGT) and the dipeptidases found in the external plasma membrane. Following plasma membrane carrier-mediated GSH release from the cell, GSH becomes accessible to the active site of γ-glutamyl transpeptidase, which catalyzes GSH breakdown into γ-glutamyl fraction and cysteinyl-glycine by transferring the γ-glutamyl fraction to an amino acid acceptor, forming γ-glutamyl-amino acid. The cysteinyl-glycine fraction is split by the enzyme dipeptidase generating cysteine and glycine. (2) The γ-glutamyl-amino acid can be metabolized to release the amino acid and 5-oxoproline, which can then be converted into glutamate to be used in the synthesis of GSH. (3) The cells incorporate cysteine and most of the intracellular cysteine is used for the synthesis of GSH. Cysteine can be used for protein synthesis and part can be degraded to sulfate and taurine. The cycle γ-glutamyl allows GSH to be used as a continuous source of cysteine. The γ-glutamyl amino acid is taken up by cells through a specific transport mechanism. Cysteinyl glycine is also taken up by cells. Inside the cell, the γ-glutamyl amino acid is hydrolyzed by γ-glutamyl cyclo-transferase and converted into oxoproline, a cyclic form of glutamate converted into glutamate via oxoprolinase, and a free amino acid.

FIGURE 6

Oxidative stress, reduced glutathione (GSH) and lung diseases. (1) Lung diseases affect alveolar cells increasing reactive oxygen species (ROS) production, reduce Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) redox regulator pathway and become defective for surfactant production. Damaged/apoptotic cells cause alveolar cell activation of nuclear factor (NF)-κB and release cytokines like interleukin (IL)-8. Alveolar type I cells augment ROS production via toll-like receptors (TLRs) 1 and 2. Inflammation enhances neutrophil extracellular trap (NET) release and increases ROS production. (2) Inflammation associated to lung diseases augments macrophage’s ROS production, inhibiting Nrf2 activation and enhancing NF-κB upregulation. ROS are counterbalanced by enzymes like superoxide dismutase (SOD), catalase (Cat), glutathione S-transferase (GST), and glutathione peroxidase (GPx) to protect cells from oxidative damage caused by nicotinamide adenine-dinucleotide phosphate (NADPH) oxidase 2 (NOX2), superoxide (O₂^–), hydrogen peroxide (H₂O₂), and myeloperoxidase (MPO). Capillary neutrophils migrate to and from alveoli by trans-endothelial (TEM) and reverse transmigration (rTEM), respectively. Inflammation can cause excessive ROS production in capillaries, red blood cell (RBC) dysfunction, thrombosis and alveolar damage. (3) Activated alveolar macrophages release increased levels of IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Inflammation-associated activated macrophages (via TLRs) reduce enzymes like SOD and Cat, among others, and activate NF-κB. NOX2 activation increases ROS production that enhance NF-κB activation. Glutathione (GSH) precursors (Cystine, cysteine, N-acetyl cysteine, NAC), and selenium (Se) restore GSH and GPx, respectively, to counteract the effects of ROS. (4) Alveolar macrophages engulf microbes and apoptotic cells via Fc (γ/α/μ) and scavenger receptors and/or pattern recognition protein receptors (PRPRs) leading to increased ROS production and cytokine release. MPO, nitric oxide (NO), O₂^–, and H₂O₂ through the Fenton and Haber-Weiss reactions that generate hydroxyl radicals, participate in ROS and RNS generation. Lung disease-associated inflammation and apoptosis [via TLRs and glycosaminoglycans (GAGs)] enhance alveolar cell ROS production that via p38MAPK, NF- κB, and AP-1 activation, contribute to epithelial injury and further inflammation. (5) Neutrophils contribute to O₂^– production, lipid peroxidation and increased oxidative stress to promote a cytokine storm (249). Administration of GSH precursors [cystine, cysteine, NAC; see (3), (4), and (5)] facilitate GSH formation to reduce oxidative stress. Abbreviations: PRRs, pattern recognition receptors; ɣ-GCS, ɣ-glutamyl cysteine synthetase; DAMPs, damage associated molecular patterns; Prxs, peroxiredoxins; NAC, N-acetyl cysteine; ɣ-GT, ɣ-glutamyl transpeptidase; PAMPs, pathogen associated molecular patterns; LPC, lysophosphatidylcholine.

FIGURE 7

Oxidative stress, reduced glutathione (GSH) and atherosclerosis. (1) Risk factors for atherosclerosis. (2) Atherosclerosis risk factors facilitate oxidative stress and inflammation in the arterial intima. Native C-reactive protein (nCRP), a pattern recognition receptor produced in the liver, macrophages, lymphocytes, smooth muscle cells (SMC), and other cells, promotes inflammation through monomeric CRP (mCRP) enhancing intimal oxidative stress. Oxidized (ox) LDL binds macrophage toll-like receptor (TLR) 4 and facilitates nicotinamide adenine dinucleotide phosphate (NADP)H oxidase 2 (Nox2) activity and superoxide (O₂^–) production causing cysteine oxidation, disulfide bridge formation and S-glutathionylation. Xanthine oxidase (XO) and inhibition of superoxide dismutase (SOD)/catalase further facilitate O₂^– cellular activity. OxLDL bound to TLRs 2 and 4 promotes foam cell formation and activates transcription factors like nuclear factor (NF)-κB facilitating cytokine storm and hyperinflammation. Excessive mitochondrial reactive oxygen species (ROS) generation further enhances cytokine production. CRP (nCRP, mCRP) can facilitate macrophage and neutrophil uptake of apoptotic cells through Fcγ and Fcα receptors, respectively (FcRs). Oxidative stress also activates the Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) redox regulator pathway in monocytes [see (3) and macrophages (2)], releasing Nrf2 to regulate the expression of genes that control antioxidant enzymes like glutathione S-transferase (GST), facilitating glutathione (GSH) activity. Macrophages, T-lymphocytes, neutrophils and SMCs can generate mCRP increasing inflammation. (3) Monocytes, macrophages, neutrophils, endothelial cells and microparticles can generate mCRP, increase O₂^– and ROS formation and reactive nitrogen species like peroxinitrite (ONOO^–), and tissue factor (TF) expression enhancing oxidation, inflammation and thrombosis. TLR 4-mediated oxLDL-binding to platelets promotes thrombosis; mCRP binding to lipid rafts and FcγRs enhances inflammation; and endothelial activation allows intimal cell migration. GSH enhancement and Nrf2 activation augment immunity and reduce atherosclerosis. (4) Foam cells and smooth muscle cells associated with atherosclerotic plaques enhance ROS formation, cytokine release and tissue factor (TF)-mediated fibrin deposition. Abbreviations: MAPK/ERK, mitogen-activated protein kinases/extracellular signal-regulated kinases; AT1R, angiotensin II type 1 receptor; PC, phosphorylcholine; LPC, lysophosphatidylcholine; MPO, myeloperoxidase; nnCRP, non-native CRP; TNF, tumor necrosis factor; IL, interleukin; ACE, angiotensin converting enzyme; MyD88/TRIF, myeloid differentiation primary response 88/TIR-domain-containing adapter-inducing interferon-β; PI3K/Akt, phosphatidylinositol-3-kinase/protein kinase B; MAPK, mitogen-activated protein kinase; AP-1, activator protein 1; CD31, cluster of differentiation 31; ICAM-1, intercellular adhesion molecule-1; Mac-1, macrophage-1 antigen; PSGL-1, P-selectin glycoprotein ligand-1; HLA-DR, human leukocyte antigen–DR isotype.

FIGURE 8

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pulmonary infection, oxidative stress and antioxidant defenses. (1) After entry of SARS-CoV-2 into the alveolus, viruses invade type II alveolar cells through angiotensin-converting enzyme 2 receptors (ACE2) and glycosaminoglycans (GAGs) [see (4)], and infected cells increase reactive oxygen species (ROS) production, reduce Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) redox regulator pathway and become defective for surfactant production. Infected cells activate nuclear factor (NF)-κB and release cytokines like interleukin (IL)-8. Alveolar type I cells augment ROS production via toll-like receptors (TLRs) 1 and 2. SARS-CoV-2 enhances neutrophil extracellular trap (NET) release and increases ROS production (2) SARS-CoV-2 augments macrophage’s ROS production, inhibiting Nrf2 activation and enhancing NF-κB upregulation. ROS are counterbalanced by enzymes like superoxide dismutase (SOD), catalase (Cat), glutathione S-transferase (GST), and glutathione peroxidase (GPx) to protect cells from oxidative damage caused by nicotinamide adenine-dinucleotide phosphate (NADPH) oxidase 2 (NOX2), superoxide (O₂^–), hydrogen peroxide (H₂O₂), and myeloperoxidase (MPO). Capillary neutrophils migrate to and from alveoli by trans-endothelial (TEM) and reverse transmigration (rTEM), respectively. SARS-CoV-2 infection can cause excessive ROS production in capillaries, red blood cell (RBC) dysfunction, thrombosis and alveolar damage. (3) Activated alveolar macrophages release increased levels of IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. SARS-CoV-2-infected macrophages (via ACE2 and TLRs) reduce enzymes like SOD and Cat, among others, and activate NF-κB. NOX2 activation increases ROS production that enhance NF-κB activation. Glutathione (GSH) precursors (Cystine, cysteine, N-acetyl cysteine, NAC), and selenium (Se) restore GSH and GPx, respectively, to counteract the effects of ROS. (4) Alveolar macrophages engulf SARS-CoV-2-infected apoptotic cells via Fc (γ/α/μ) and scavenger receptors and/or pattern recognition protein receptors (PRPRs) leading to increased ROS production and cytokine release. (5) Neutrophils contribute to O₂^– production, lipid peroxidation and increased oxidative stress to promote the cytokine storm. Abbreviations: TMPRSS2, Transmembrane protease Serine 2; PRPs, pattern recognition proteins. Reprinted from Labarrere and Kassab (335).

FIGURE 9

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enhances oxidative stress and atherosclerosis progression. (1) SARS-CoV-2 structure. (2) SARS-CoV-2 viruses facilitate oxidative stress and inflammation in the arterial intima. Native C-reactive protein (nCRP), a marker of severe SARS-CoV-2 produced in liver, macrophages, lymphocytes, smooth muscle cells (SMC) and other cells, promotes inflammation through monomeric CRP (mCRP) enhancing intimal oxidative stress. SARS-CoV-2 binds macrophage toll-like receptor (TLR) 4 and facilitates nicotinamide adenine dinucleotide phosphate (NADP)H oxidase 2 (Nox2) activity and superoxide (O₂^–) production causing cysteine oxidation, disulfide bridge formation and S-glutathionylation. Xanthine oxidase (XO) and inhibition of superoxide dismutase (SOD)/catalase further facilitate O₂^– cellular activity. SARS-CoV-2 can bind TLRs 2 and 4 and activate transcription factors like nuclear factor (NF)-κB facilitating cytokine storm and hyperinflammation. Excessive mitochondrial reactive oxygen species (ROS) generation further enhances cytokine production. CRP (nCRP, mCRP) can facilitate macrophage and neutrophil uptake of SARS-CoV-2-infected apoptotic cells through Fcγ and Fcα receptors, respectively (FcRs). Oxidative stress also activates the Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) redox regulator pathway in monocytes [see (3) and macrophages (2)], releasing Nrf2 to regulate the expression of genes that control antioxidant enzymes like glutathione S-transferase (GST), facilitating glutathione (GSH) activity. Macrophages, T-lymphocytes, neutrophils and SMCs can generate mCRP increasing inflammation. (3) Monocytes, macrophages, neutrophils, endothelial cells and microparticles can generate mCRP, increase superoxide (O₂^–) and ROS formation and reactive nitrogen species like peroxinitrite (ONOO–), and tissue factor (TF) expression enhancing oxidation, inflammation and thrombosis. TLR 4-mediated SARS-CoV-2-binding to platelets promotes thrombosis, mCRP binding to lipid rafts and FcγRs enhances inflammation and endothelial activation allows intimal cell migration. (4) Foam cells and smooth muscle cells associated with atherosclerotic plaques enhance ROS formation, cytokine release and tissue factor (TF)-mediated fibrin deposition. Abbreviations: MAPK/ERK, mitogen-activated protein kinases/extracellular signal-regulated kinases; AT1R, angiotensin II type 1 receptor; PC, phosphorylcholine; LPC, lysophosphatidylcholine; MPO, myeloperoxidase; nnCRP, non-native CRP; TNF, tumor necrosis factor; IL, interleukin; ACE, angiotensin converting enzyme; MyD88/TRIF, myeloid differentiation primary response 88/TIR-domain-containing adapter-inducing interferon-β; PI3K/Akt: phosphatidylinositol-3-kinase/protein kinase B; MAPK, mitogen-activated protein kinase; AP-1, activator protein 1; CD31, cluster of differentiation 31; ICAM-1, intercellular adhesion molecule-1; Mac-1, macrophage-1 antigen; PSGL-1, P-selectin glycoprotein ligand-1; HLA-DR, human leukocyte antigen–DR isotype. Reprinted from Labarrere and Kassab (335).