NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Glutathione

Glutathione (GSH) is a ubiquitous low molecular weight thiol tripeptide containing glutamate, cysteine, and glycine (Glu-Cys-Gly) discovered by Hopkins, Hunter and Eagles in the 1920s . Nucleophylic properties making glutatione as a major component of redox buffer in the cells. Glutathione-dependent enzymes such as glutatione peroxidases (GPXs), glutaredoxins (GRXs) and glutatione S-transferases (GSTs) are typical for most living organisms. The intracellular concentration of GSH is in ranges ofapproximately 0.1 to 15 mM and GSH/GSSH ratio depends on cellular compartment. GSH work reducing agent for disulfides non-enzymatically or enzymatically (in glutaredoxing cycle). GSSG work as major thiol modifying agent in glutatione S-transferases cycle [ 1, 2, 3, 4, 5 ].

Glutathione is form the largest intracellular pools of non-protein diothiol reducing equivalents and has a critical role in regulating redox environments. GSH/GSSG rationis highly responsive to the external influences and serves to protect cellular macromolecules from oxidative damage and maintains cellular redox homeostasis. The major oxidized form of glutathione, GSSG, represents two glutathione molecules cross-linked via a disulfide bond [ 5, 6, 7 ].

The biosynthesis of glutatione in cells is accomplished by six enzymes found on the plasma membrane and in the cytosol from the precursor amino acids Glu, Cys, and Gly. Extracellular GSH is selectively transported into the cell but somer of tissues requiring degradation to single amino acids before transport into the cell. The universal facilitator of GSH breakdown and only know glutathionase enzyme is γ-glutamyltranspeptidase (GGT) which is located on the outer leaf of the cell's plasma membrane. γ-glutamyltranspeptidase cleaves glutathione at the unique γ-glutamyl bond to form free glutamate and the dipeptide Cys-Gly, which is degraded to Cys and Gly for transport in to cells via amino acid transporters. The Glu is quickly reunited with another amino acid (usually a Cys) and transported into the cell as γ-Glu-amino acid. Once inside the cell, precursor amino acids are enzymatically processed and passed along to enzymes responsible for GSH synthesis beginning with cyclization and decyclization reactions to release the bound amino acid and present free Glu as the first substrate precursor for GSH biosynthesis. Glu and Cys are combined in to form γ-glutamylcysteine using the enzyme glutamate-cysteine ligase (GCL). The last step of GSH biosynthesis involve the addition of Gly by ATP and Mg2+ dependent glutathione synthase (GS). [ 7, 8, 9, 10, 11, 12 ].

The γ-glutamyl cycle (GCC) and de novo synthesis of glutathione. As GSH is usually not directly transported inside the cell, GSH breakdown into components more amenable to transport can occur. Once precursors are available intracellularly, GSH can be resynthesized to regenerate GSH via de novo synthesis enzymes (glutamate-cysteine ligase and glutathione synthetase) [ 7, ].

Canonical γ-glutamyl cycle [ 11, ].

Glutathione does not react nonenzymaticaly with hydroperoxides, hovewer, it is a component of glutathione peroxidase catalytic cycle, thus involved in reduction of H2O2 and lipid hydroperoxides. Not all glutathione peroxidase utilize glutathione and some of them have specificity for thioredoxin or other thiol oxidoreductases. The reduction of hydroperoxides by glutathione peroxidase involves the conversion of the active-site Sec or Cys residue to the selenenic or sulfenic acid intermediate (-Se-OH or -S-OH), reduction of such intermediate by GSH, leading to (-Se-SG) or thiol (-S-SG) intermediate [ 13, 14 ]. As next step, this intermediate reacts with a second GSH molecule restoring active site Sec or Cys and producing oxidized glutathione (GSSG). GSSG is subsequently reduced by glutathione reductase (GR), which uses NADPH as a source of reducing equivalents [ 15, 16, 17 ].

H2O2 +2GSH + Glutathione Peroxidase → 2H2O + GSSG ;
ROOH + 2GSH + Glutathione Peroxidase (GPX) → 2ROH + GSSG ;

Glutatione itself is not efficient reductant of disufides, hovewer, it is involved in providing of redox equivalent to glutaredoxins and work as component of glutaredoxins catalytic cycle. IThe electrons are transferred from NADPH to glutathione reductase, then to glutathione and then to glutaredoxin, resulting in the GRX-mediated reduction of disulfides in target proteins [ 18, 19 ]:

2GSH + Protein-(S)2 + Glutareoxin (GRX) → GSSG + Protein-(SH)2 ;
GSSG + NADPH + H+ Glutathione Reductase →NADP+ + GSH ;

Protein glutathionylation is a S-thiolation of protein cysteines by glutathione. Glutathionylaton protects proteins in oxidative streess conditions and is involved in regulation of protein function. Glutathionylated proteins can be formed either by direct oxidation or by thiol-disulfide exchange mechanism [ 12, 21 ]:

Protein-SH + GSH + Oxidant ⇆ Protein-SSG ;
Protein-SH + GSSG ⇆ Protein-SSG + GSH ;

Another glutathionylation mechanisms involve formation of S-nitrosothiols and sulfenic acid [ 12 ]:

Protein-SNO + GSH → Protein-SSG + HNO ;
Protein-SH + GSNO → Protein-SSG + HNO ;
Protein-SOH + GSH → Protein-SSG + H2O ;

Glutathionylated proteins can be reduced to free thiols by the reverse of the thiol-disulfide exchange reaction when the GSH/GSSG ratio is high, or reduction can be catalysed by thiol disulfide oxidoreductases [ 12, 21 ].

Glutathione can forms conjugates with wide spectrum of electrophilic compounds nonenzymatically, when the electrophile is very reactive, or through the action of glutathione S-transferases (GST). Conjugation with clutathione is essential process for xenobiotic and normal physiological metabolism: [ 12, 21, 22, 23, 24 ]:

GSH + Electrophilic Compounds + Glutathione S-transferase (GST) ⇆ GS-(Electrophilic Compounds) Conjugate ;

Another role of glutathione in reactive oxygem spicies detoxification is based on its ability to react with carbon centered radicals [ 12, 21, 25 ]:

R.- + GSH → RH + GS.- ;
GS.- + GS- → GSSG.- ;
GSSG.- + O2 → GSSG + O2.- ;

The major ammount of glutathione is retained in the cytoplasm, hovewer, glutathione is also used in other cellular organelles such as nucleus, mitochondria, chloroplast (in plants), endoplasmic reticulum, vacuoles and the extracellular space. Each organelle differs has diffrerent absolute concentrations of glutathione, and oxidized to reduced glutathione ratios. Glutathione is also effluxed out of the cell into the extracellular space medium [ 26, 27, 28, 29 ].

Known and predicted glutathione transporters in the yeast S. cerevisiae [ 26, ].

Hgt1p in yeast S. cerevisiae was the first identified high-specificityand high-affinity glutathione transporter. Hgt1p belongs to oligopeptide transporter family which was found in fungi, plants and prokaryotes but not in mammals. This transporter is required for the uptake of glutathione from the extracellular medium [ 30, 31 ].

MFS family chloroquine resistance transporter (PfCRT) is function as glutathione transporters in plants. Three genes have been CRT-like proteins were found to localize to the chloroplast and were responsible for glutathione efflux from the chloroplast to the cytoplasm [ 32 ]. Glutathione uptake pathways in bacteria are different between organisms and several different transport pathways is known YliABCD operon is involved in GSH transport in E. coli. The proteins encoded by the genes yliA, yliB, yliC, and yliD form a multi-subunit protein belonging to the periplasmic binding-protein-dependent ABC transporter family in prokaryotes [ 26, 33 ]. The gram-negative bacteria Haemophilus influenzae is a natural cysteine and glutathione auxotroph and acquires glutathione using by the dipeptide permease DppBCDF complex [ 26, 34 ].

Gex1p and its paralogue Gex2p are vacuolar and plasma membrane glutathione efflux pumps were predicted as glutathione/proton antiporters. Gxa1p is a glutathione exporter that belongs to ABC transporter family.

Two vacuolar localized MRPs in yeasts have been shown to be able to function in low affinity glutathione transport. These are the yeast cadmium factor-1 (Ycf1p) and the bile pigment transporter-1 (Bpt1p). These proteins, though primarily recognized as glutathione-conjugate ABC-dependent efflux pumps.

Glutathione efflux in bacteria is mediated by a heterodimeric ABC-type transporter CydDC, which is homologous to the mammalian CFTR pump. CydDC is crucial for maintenance of the redox-homeostasis in the periplasm.

Mammalian glutathione transporters:

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