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GLUTATHIONE SYNTHESIS
Shelly C. Lu, M.D.*
Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases,
Southern California Research Center for ALPD and Cirrhosis, Keck School of Medicine USC, Los
Angeles, CA 90033
Abstract
BACKGROUND—Glutathione (GSH) is present in all mammalian tissues as the most abundant
non-protein thiol that defends against oxidative stress. GSH is also a key determinant of redox
signaling, vital in detoxification of xenobiotics, regulates cell proliferation, apoptosis, immune
function, and fibrogenesis. Biosynthesis of GSH occurs in the cytosol in a tightly regulated
manner. Key determinants of GSH synthesis are the availability of the sulfur amino acid
precursor, cysteine, and the activity of the rate-limiting enzyme, glutamate cysteine ligase (GCL),
which is composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The second enzyme of
GSH synthesis is GSH synthetase (GS).
SCOPE OF REVIEW—This review summarizes key functions of GSH and focuses on factors
that regulate the biosynthesis of GSH, including pathological conditions where GSH synthesis is
dysregulated.
MAJOR CONCLUSIONS—GCL subunits and GS are regulated at multiple levels and often in a
coordinated manner. Key transcription factors that regulate the expression of these genes include
NF-E2 related factor 2 (Nrf2) via the antioxidant response element (ARE), AP-1, and nuclear
factor kappa B (NFκB). There is increasing evidence that dysregulation of GSH synthesis
contributes to the pathogenesis of many pathological conditions. These include diabetes mellitus,
pulmonary and liver fibrosis, alcoholic liver disease, cholestatic liver injury, endotoxemia and
drug-resistant tumor cells.
GENERAL SIGNIFICANCE—GSH is a key antioxidant that also modulates diverse cellular
processes. A better understanding of how its synthesis is regulated and dysregulated in disease
states may lead to improvement in the treatment of these disorders.
Keywords
GSH; glutamate-cysteine ligase; GSH synthase; Nrf2; MafG; c-Maf; antioxidant response element
1. Introduction
Glutathione (GSH) is a tripeptide, γ-L-glutamyl-L-cysteinylglycine, present in all
mammalian tissues at 1–10 mM concentrations (highest concentration in liver) as the most
abundant non-protein thiol that defends against oxidative stress. GSH is also a key
© 2012 Elsevier B.V. All rights reserved.
*To whom correspondence should be sent: HMR Rm 415, Department of Medicine, Keck School of Medicine USC, 2011 Zonal Ave.,
Los Angeles, CA 90033, Tel: (323) 442-2441, FAX: (323) 442-3234, shellylu@usc.edu.
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Author Manuscript
Biochim Biophys Acta
. Author manuscript; available in PMC 2014 May 01.
Published in final edited form as:
Biochim Biophys Acta
. 2013 May ; 1830(5): 3143–3153. doi:10.1016/j.bbagen.2012.09.008.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
determinant of redox signaling, vital in detoxification of xenobiotics, modulates cell
proliferation, apoptosis, immune function, and fibrogenesis. This review is focused on
factors that determine GSH synthesis and pathologies where dysregulation in GSH synthesis
may play an important role with emphasis on the liver. This is because the liver plays a
central role in the interorgan GSH homeostasis [1].
2. Structure and functions of GSH
GSH exists in the thiol-reduced and disulfide-oxidized (GSSG) forms [2]. GSH is the
predominant form and accounts for >98% of total GSH [3–5]. Eukaryotic cells have three
major reservoirs of GSH. Most (80–85%) of the cellular GSH are in the cytosol, 10–15% is
in the mitochondria and a small percentage is in the endoplasmic reticulum [6–8]. Rat liver
cytosolic GSH turns over rapidly with a half-life of 2–3 hours. The structure of GSH is
unique in that the peptide bond linking glutamate and cysteine of GSH is through the γ-
carboxyl group of glutamate rather than the conventional α-carboxyl group. The only
enzyme that can hydrolyze this unusual bond is γ-glutamyltranspeptidase (GGT), which is
only present on the external surfaces of certain cell types [9]. As a consequence, GSH is
resistant to intracelluar degradation and is only metabolized extracellularly by cells that
express GGT. This allows for released GSH to be broken down and its constituent amino
acids taken up by cells and reincorporated into GSH (so called γ-glutamyl cycle, see below).
The bulk of plasma GSH originates from the liver, which plays a central role in the
interorgan homeostasis of GSH by exporting nearly all of the GSH it synthesizes into
plasma and bile [1,10,11]. Thus, dysregulation of hepatic GSH synthesis has impact on GSH
homeostasis systemically.
GSH serves several vital functions including antioxidant defense, detoxification of
xenobiotics and/or their metabolites, regulation of cell cycle progression and apoptosis,
storage of cysteine, maintenance of redox potential, modulation of immune function and
fibrogenesis [4, 5,9,12–15]. Some of these key functions, namely antioxidant defense, redox
signaling, storage of cysteine via the γ-glutamyl cycle, regulation of growth and death are
described in more detail below.
2.1 Antioxidant function of GSH
The antioxidant function of GSH is accomplished largely by GSH peroxidase (GPx)-
catalyzed reactions, which reduce hydrogen peroxide and lipid peroxide as GSH is oxidized
to GSSG. GSSG in turn is reduced back to GSH by GSSG reductase at the expense of
NADPH, forming a redox cycle [13]. Organic peroxides can also be reduced by GPx and
GSH S-transferase. Catalase can also reduce hydrogen peroxide but it is present only in
peroxisome. This makes GSH particularly important in the mitochondria in defending
against both physiologically and pathologically generated oxidative stress [16,17]. As GSH
to GSSG ratio largely determines the intracellular redox potential (proportional to the log of
[GSH]2/[GSSG]) [5], to prevent a major shift in the redox equilibrium when oxidative stress
overcomes the ability of the cell to reduce GSSG to GSH, GSSG can be actively exported
out of the cell or react with a protein sulfhydryl group leading to the formation of a mixed
disulfide. Thus, severe oxidative stress depletes cellular GSH [13] (Figure 2).
2.2 GSH in redox signaling
GSH regulates redox-dependent cell signaling. This is largely accomplished by modifying
the oxidation state of critical protein cysteine residues [5,18]. GSH can be reversibly bound
to the –SH of protein cysteinyl residues (Prot-SH) by a process called glutathionylation,
generating glutathionylated proteins (Prot-SSG), which can either activate or inactivate the
protein [18]. This is a mechanism to protect sensitive protein thiols from irreversible
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oxidation and may also serve to prevent loss of GSH under oxidative conditions (Figure 2).
Deglutathionylation can then occur through glutaredoxin and sulfiredoxin-catalyzed
reactions using GSH as a reductant [15]. Many transcription factors and signaling molecules
have critical cysteine residues that can be oxidized and this is an important mechanism
whereby reactive oxygen and nitrogen species (ROS and RNS) regulate protein function and
cell signaling that can be modulated by GSH [13,15].
2.3 GSH and the γ-glutamyl cycle
Alton Meister first described the γ-glutamyl cycle in the early 1970’s, which allows GSH to
serve as a continuous source of cysteine [19] (Figure 3). This is an important function as
cysteine is extremely unstable and rapidly auto-oxidizes to cystine extracellularly, which can
generate potentially toxic oxygen free radicals [19]. In the γ-glutamyl cycle, GSH is
released from the cell and the ecto-enzyme GGT transfers the γ-glutamyl moiety of GSH to
an amino acid (the best acceptor being cystine), forming γ-glutamyl amino acid and
cysteinylglycine. The γ-glutamyl amino acid can be transported back into the cell and once
inside, the γ-glutamyl amino acid can be further metabolized to release the amino acid and
5-oxoproline, which can be converted to glutamate and used for GSH synthesis.
Cysteinylglycine is broken down by dipeptidase to generate cysteine and glycine. Most cells
readily take up cysteine. Once taken up, the majority of cysteine is incorporated into GSH,
some is incorporated into protein, and some is degraded into sulfate and taurine [19].
2.4 GSH regulates growth and death
In many normal and malignant cell types, increased GSH level is associated with a
proliferative response and is essential for cell cycle progression [20–26]. In normal
hepatocytes, GSH level increases when cells shift from G0 to G1 phase of the cell cycle
in
vitro
[25], and after 2/3 partial hepatectomy prior to the onset of increased DNA synthesis
[27]. If this increase in GSH was blocked, DNA synthesis following partial hepatectomy
was reduced by 33% [26]. In liver cancer and metastatic melanoma cells, GSH status also
correlated with growth [26,28]. Interestingly, hepatocyte growth factor (HGF) induces the
expression of GSH synthetic enzymes and acts as a mitogen in liver cancer cells only under
subconfluent cell density condition and the mitogenic effect required increased GSH level
[29]. A key mechanism for GSH’s role in DNA synthesis relates to maintenance of reduced
glutaredoxin or thioredoxin, which are required for the activity of ribonucleotide reductase,
the rate-limiting enzyme in DNA synthesis [30]. In addition, the GSH redox status can affect
the expression and activity of many factors important for cell cycle progression. Of
particular interest is the finding that GSH co-localizes to the nucleus at the onset of
proliferation, which through redox changes can affect the activity of many nuclear proteins
including histones [14,31]. These recent studies show that a reducing condition in the
nucleus is necessary for cell cycle progression [14].
GSH also modulates cell death. Apoptosis, characterized by chromatin condensation,
fragmentation and internucleosomal DNA cleavage, and necrosis, characterized by rupture
or fragmentation of the plasma membrane and ATP depletion [32] can coexist and share
common pathways, such as involvement of the mitochondria [33]. GSH modulates both
types of cell death. GSH levels influence the expression/activity of caspases and other
signaling molecules important in cell death [4,32]. GSH levels fall during apoptosis in many
different cell types, due to ROS, enhanced GSH efflux, and decreased GCL activity (see
section on post-translational regulation of GCLC) [34,35]. Although GSH efflux may be a
mechanism to circumvent the normally protective role of GSH, it appears essential for
apoptosis to occur in many cell types [4, 36]. However, profound GSH depletion can convert
apoptotic to necrotic cell death [34], suggesting very high levels of ROS may overwhelm the
apoptotic machinery. Consistently, severe mitochondrial GSH depletion leads to increased
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levels of ROS and RNS, mitochondrial dysfunction and ATP depletion, converting apoptotic
to necrotic cell death [32].
3. Synthesis of GSH
The synthesis of GSH from its constituent amino acids involves two ATP-requiring
enzymatic steps: formation of γ-glutamylcysteine from glutamate and cysteine and
formation of GSH from γ-glutamylcysteine and glycine (Figure 1). The first step of GSH
biosynthesis is rate limiting and catalyzed by GCL (EC 6.3.2.2; formerly γ-glutamylcysteine
synthetase), which is composed of a heavy or catalytic (GCLC, Mr ~ 73 kDa) and a light or
modifier (GCLM, Mr ~ 31 kDa) subunit, which are encoded by different genes in fruit flies,
rodents and humans [37–41]. In contrast, GCL in yeast and bacteria have only a single
polypeptide [41]. GCLC exhibits all of the catalytic activity of the isolated enzyme and
feedback inhibition by GSH [42]. GCLM is enzymatically inactive but plays an important
regulatory function by lowering the Km of GCL for glutamate and raising the Ki for GSH
[38,43]. Thus, the holoenzyme is catalytically more efficient and less subject to inhibition by
GSH than GCLC. However, GCLC alone does have enzymatic activity as
Gclm
knockout
mice are viable but have markedly reduced tissue GSH levels (reduced by about 85 to 90%)
[44]. Redox status can influence GCL activity via formation of the holoenzyme [45]. Most
of the GCL holoenzyme can be reversibly dissociated by treatment with dithiothreitol [42],
while oxidative stress may enhance holoenzyme formation as it increases GCL activity in
the absence of any change in the expression of GCL subunits [45].
Under physiological conditions GCL is regulated by: (a) nonallosteric feedback competitive
inhibition (with glutamate) by GSH (Ki=2.3mM) [46] and (b) availability of L-cysteine [9].
The Km values of GCL for glutamate and cysteine are 1.8 and 0.1–0.3 mM, respectively
[46]. The intracellular glutamate concentration is 10-fold higher than the Km value but
cysteine concentration approximates the apparent Km value [47].
The second step in GSH synthesis is catalyzed by GSH synthetase (GS, EC 6.3.2.3, also
known as GSH synthase). GS is composed of two identical subunits (Mr ~ 118 kDa) and is
not subject to feedback inhibition by GSH [48]. Since the product of GCL, γ-
glutamylcysteine, is present at exceedingly low concentrations when GS is present, GCL is
considered rate limiting [41]. In support of this is the finding that overexpression of GS
failed to increase GSH level whereas overexpression of GCL increased GSH level [49].
Although GS is generally thought not to be important in the regulation of GSH synthesis,
there is accumulating evidence that GS is important in determining overall GSH synthetic
capacity in certain tissues and/or under stressful conditions [13]. Surgical trauma decreased
GSH levels and GS activity in skeletal muscle while GCL activity was unchanged [50]. In
rat hepatocytes, increased GS expression further enhanced GSH synthesis above that
observed with increased GCLC expression alone [51].
3.1 Factors that determine cysteine availability
Cysteine is derived normally from the diet, protein breakdown and in the liver, from
methionine via transsulfuration (see below). Cysteine is unstable extracellularly where it
readily autoxidizes to cystine, which is taken up by some cells and is rapidly reduced to
cysteine intracellularly [47]. In hepatocytes, the key factors that regulate cysteine
availability include membrane transport of cysteine (via the ASC system), cystine (via the
Xc- system which is induced under oxidative stress), methionine (via the L system) and the
activity of the transsulfuration pathway [47,52,53].
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3.2 Transsulfuration pathway
The transsulfuration pathway (also called the cystathionine pathway) allows the utilization
of methionine for GSH synthesis [54] (Figure 4). Liver plays a central role in methionine
metabolism as up to half of the daily intake of methionine is catabolized in the liver. The
first step in methionine catabolism is the generation of S-adenosylmethionine (SAMe), the
principal biological methyl donor, in a reaction catalyzed by methionine adenosyltransferase
(MAT) [55]. Under normal conditions, most of the SAMe generated is used in
transmethylation reactions [56]. SAMe donates its methyl group to a large variety of
acceptor molecules in reactions catalyzed by methyltransferases (MTs), generating S-
adenosylhomocysteine (SAH), which is in turn hydrolyzed to homocysteine (Hcy) and
adenosine through a reversible reaction catalyzed by SAH hydrolase. SAH is a potent
competitive inhibitor of methylation reactions so that prompt removal of Hcy and adenosine
is required to prevent SAH accumulation. In hepatocytes, Hcy can be remethylated to
generate methionine by methionine synthase (MS), which requires normal levels of folate
and vitamin B12, and betaine homocysteine methyltransferase (BHMT), which requires
betaine. Hcy can also be converted to cysteine via the transsulfuration pathway by a two-
enzyme process. First, Hcy condenses with serine to form cystathionine in a reaction
catalyzed by cystathionine β synthase (CBS), which requires vitamin B6. The second step
cleaves cystathionine, catalyzed by another vitamin B6-dependent enzyme γ-cystathionase,
and releases free cysteine for GSH synthesis [56]. The transsulfuration pathway is
particularly active in hepatocytes but outside of the liver, it is either absent or present at very
low levels [56]. The hepatic transsulfuration pathway activity is markedly impaired or
absent in the fetus and newborn infant and in cirrhotic patients [13]. Part of the mechanism
relates to decreased cofactor availability (such as B vitamins). In addition, cirrhotic patients
also have decreased MAT activity and diminished SAMe biosynthesis, which further
contribute to decreased GSH levels [55].
4. Regulation of glutamate-cysteine ligase (GCL)
Changes in GCL activity can result from regulation at multiple levels affecting only GCLC
or both GCLC and GCLM.
4.1 GCLC pre-translational regulation
Many conditions are known to affect GCLC pre-translationally. Drug-resistant tumor cell
lines and oxidative stress are associated with increased cell GSH levels, GCL activity,
GCLC mRNA levels and GCLC gene transcription [57–63]. While many of these treatments
induced both GCLC and GCLM expression, selective transcriptional induction of only
GCLC occurred when cultured rat hepatocytes were treated with insulin or hydrocortisone
[64–66]. The physiologic significance of the hormonal effect was confirmed using insulin-
deficient diabetic or adrenalectomized rats. Both exhibited lower hepatic GSH levels and
GCL activity, which were prevented with hormone replacement [64]. Importantly, lower
levels of GSH in the erythrocytes of diabetic patients and increased susceptibility to
oxidative stress of these cells have been reported [67]. Kim et al reported that the effect of
insulin on GSH levels and GCLC expression in rat hepatocytes involve PI3K/Akt/p70S6K
but not ERK, JNK and p38 MAPK [68]. However, Li et al reported that insulin’s effect on
GSH synthesis in cardiac myocytes required PI3K, MEK and p38 MAPK [69]. More
recently, Langston et al reported that in human brain endothelial cell line, insulin activated
GCLC promoter activity under altered glycemic condition (both low and high) that required
PI3K/Akt/mTOR signaling [70]. Thus, while the PI3K signaling pathway appears to be a
central player in mediating insulin’s effect in many cell types, other signaling pathways
activated by insulin may act more in a tissue-specific manner in the up-regulation of GCLC
expression and GSH level.
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Another condition where GCLC is induced transcriptionally while GCLM is unchanged is
during rapid liver growth. Plating hepatocytes under low-density and liver regeneration
following partial hepatectomy are such examples [25,27,65]. Increased GSH levels and
GCLC transcription and mRNA levels (GCLM expression was unchanged) also occur in
human hepatocellular carcinoma (HCC) [26]. Thus, hormones and increased growth
selectively regulate hepatic GCLC expression in most circumstances. Since an increase in
GCLC expression alone led to an increase in GSH level, we speculated that GCLC might be
limiting in hepatocytes. However, this point remains controversial, as others have reported
the opposite, namely GCLM is limiting [13,45].
Transforming growth factor-β1 (TGF-β1), a pleiotropic cytokine implicated in the
pathogenesis of idiopathic pulmonary fibrosis and in liver fibrosis, has also been shown to
regulate GSH synthesis at the level of GCLC [71–73]. In type II alveolar epithelial cells,
TGF-β1 lowered the transcriptional activity of GCLC [72]. Similarly, in rat hepatic stellate
cells (HSCs) TGF-β1 suppressed the expression of GCLC (no effect on GCLM) and
lowered GSH levels [73]. This was a key mechanism for TGF-β1-mediated profibrogenic
effect in HSCs that is targeted by (−)-epigallocatechin-3-gallate, the major constituent of
green tea that exerts antioxidant effect [73].
Other conditions known to influence expression of GCLC at the transcriptional level include
treatments with antioxidants such as butylated hydroxyanisole [74,75], 5,10-dihydroindeno
[1,2-b]indole and tert-butyl hydroquinone (TBH) [44,76,77], inducers of Phase II
detoxifying enzymes such as β-naphthoflavone (β-NF) [78], formation of Michael reaction
acceptors (containing an electrophilic electron-deficient center that is susceptible to
nucleophilic attack) by treatment with diethyl maleate (DEM) to produce GSH conjugates
[75], heat shock [79], zinc [80], melatonin [81], curcumin [82], and lipid peroxidation
products such as 4-hydroxynonenal (4-HNE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2)
[83–86], trivalent arsenite (As3+) [87], and ajoene, a major compound in garlic extracts [88].
4-HNE is a major end product of lipid peroxidation that is present in normal human plasma
at concentrations ranging from 0.1μM to 1.4μM and this can increase more than 10 folds
during oxidative stress
in vivo
[84]. 4-HNE was shown to induce GCLC with concentrations
found in human plasma, suggesting that the “basal” GCLC expression is under regulation by
products of lipid peroxidation [84]. In endothelial cells, nitric oxide (NO) was found to
protect against H2O2-induced toxicity via induction of GCLC that required the participation
of zinc and Nrf2 [89]. Thus, GCLC gene expression is up-regulated when increased cellular
defense is needed. However, if the toxic or injurious insult persists, GCLC expression may
become dysregulated. An example is treatment with the toxic bile acid lithocholic acid,
which caused initial GCLC induction, followed later by suppression at the transcriptional
level in hepatocytes [90]. A similar pattern also occurred during bile duct ligation (BDL)
[90] (see below under GSH synthesis dysregulation during cholestatic liver injury).
There have been numerous studies that examined the molecular mechanism(s) of GCLC
transcriptional regulation [13]. Rodents and human GCLC promoter regions share similar
regulatory mechanisms. The promoter region of GCL subunits of human, rat, and mouse has
been cloned [78,91–97]. Consensus NFκB, Sp-1, activator protein-1 (AP-1), AP-2, metal
response (MRE), and ARE/EpRE elements have been identified in the human GCLC
promoter. A proximal AP-1 element (−263 to −269) was found to be critical in mediating
the effect of oxidative stress-induced increase in human GCLC transcription [98–103].
However, a distal ARE element located ~ 3.1kb upstream of the transcriptional start site of
human GCLC was found to mediate constitutive and β-NF inducible expression in HepG2
cells (a human hepatoma cell line) [78]. The transcription factor Nrf2, possibly in complexes
with other Jun or Maf proteins, was found to be responsible for
trans
-activating the human
GCLC promoter via binding to ARE4 in response to numerous treatments including β-NF,
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pyrrolidine dithiocarbamate, TBH [104,105], and insulin [106]. The ARE4 is also the site of
TGF-β1 action in type II alveolar cells, as TGF-β1 induced the binding of Fra-1/c-Jun dimer
to the internal AP-1 sequence of the ARE4 site that led to suppression of GCLC
transcriptional activity [72]. Thus, depending on the make up of the transcription factors
bound to the ARE, opposite effects may occur. Indeed, this is further illustrated in
cholestatic liver injury (see below). Thus, ARE appears to be the key element for various
inducers of GCLC. However, As3+, despite being an inducer of oxidative stress, induced
GCLC transcriptional activity by a mechanism that is independent of Nrf1 or Nrf2 [87]. The
transcription factor(s) responsible for As3+-mediated GCLC induction is unknown.
Nrf2 is the key transcription factor required for activation of ARE. Nrf1 and Nrf2 are
members of the cap ‘n’ collar-basic leucine zipper proteins (CNC-bZIP) and both can
trans
-
activate ARE [107–109]. Under non-stressful physiological condition, Nrf2 is kept in the
cytosol by Keap1, a component of an E3 ubiquitin ligase complex that targets Nrf2 for
proteasomal degradation [110,111]. Oxidative stress and treatment with many agents that
activate Nrf2 act by modifying either Keap1 or Nrf2 posttranslationally to cause their
dissociation [88,110]. Once Nrf2 is released from Keap1, it escapes proteasomal degradation
and translocates to the nucleus to induce genes involved in antioxidant defense [110]. NO’s
activation of Nrf2 in endothelial cells required an increase in free zinc level intracellularly
but how zinc activates Nrf2 is not clear [89]. Once Nrf2 translocates to the nucleus, it forms
heterodimers with small Maf (MafG, MafK and MafF) and Jun (c-Jun, Jun-D, and Jun-B)
proteins to bind to ARE [108]. Nrf2/MafG heterodimer generally activates ARE-dependent
gene transcription and has also been reported to enhance Nrf2 nuclear retention [108,112].
Another key mechanism that controls Nrf2 nuclear level is importin α7-mediated Keap1
nuclear import [111]. Thus, Keap1 shuttles between the nucleus and the cytoplasm and the
nuclear import requires the interaction of importin α7 with the C-terminal Kelch domain of
Keap1 [111]. Keap1 contains a strong nuclear export signal, which facilitates the nuclear
export of Keap1-Nrf2 complex to keep Nrf2 activation and ARE-dependent gene induction
under control [111]. Keap1 does not control Nrf1’s activity; instead Nrf1 is primarily
localized to the membrane of the endoplasmic reticulum and is released and translocates to
the nucleus during endoplasmic reticulum stress [113]. Nrf1 knockout mice die
in utero
but
fetal hepatocytes and embryonal fibroblasts have lower GSH levels and are more susceptible
to oxidative stress [107,114]. Nrf2 knockout mice also exhibit lower GSH levels and are
more susceptible to acetaminophen-induced liver injury [115]. Both Nrf1 and Nrf2 knockout
mice have lower GCLC expression [114,115] and overexpression of Nrf1 and Nrf2 can
induce the human GCLC promoter activity [104,116]. Nrf1 and Nrf2 may induce GCLC
promoter directly and indirectly. Although the rat GCLC promoter also contain a distal ARE
4 kb upstream [117], the 1.8 kb 5′-flanking region of the rat GCLC does not contain any
consensus ARE element (5′-G/ATG/TAG/CNNNGCA/G-3′) [105] and yet TBH treatment
can induce the reporter activity driven by this 1.8 kb construct [118]. The explanation lies in
cross talks between Nrf1/Nrf2 and AP-1 and NF B family members. The basal expression
and nuclear binding activities of c-Jun, c-Fos, p50 and p65 are lower in fibroblast cells
lacking Nrf1 or Nrf2. Other AP-1 and NF B family members are either unaffected (JunB,
JunD) or increased (Fra-1, JAB1, and c-Rel). Overexpression of Nrf1 and Nrf2 restored the
rat 1.8 kb-GCLC promoter activity and response to TBH by enhancing the expression of key
NFκB and AP-1 family members [118]. However, this was blocked if the AP-1 and NFκB
binding sites were mutated, proving the importance of these
cis
-acting elements at least in
the rat GCLC. Interactions between NF B and AP-1 also occur [97]. Tumor necrosis factor
α (TNFα) induces NFκB and AP-1 nuclear-binding activities and both are required for
normal expression of both GCL subunits and GS in rat. While all three genes have multiple
AP-1-binding sites, only GCLC has an NFκB-binding site. The explanation for the ability of
NFκB to induce rat GCLM and GS promoter activity is that NFκB can increase AP-1
expression and nuclear binding activity. Thus, both c-Jun and NFκB are required for basal
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and TNFα-mediated induction of GSH synthetic enzymes in H4IIE cells (a rat hepatoma
cell line). While NFκB may exert a direct effect on the GCLC promoter, it induces the
GCLM and GS promoters indirectly via c-Jun. These findings further illustrate the complex
cross talks among the different families of transcription factors. In mouse, both GCLC and
GCLM have NFκB binding sites and the basal expression of these two genes requires NFκB
[119].
In addition to ARE/EpRE, AP-1 and NFκB, c-Myc has been identified to also contribute to
the basal expression and induction of human GCL under oxidative stress [120]. Thus, down-
regulation of c-Myc lowered GSH while overexpression of c-Myc increased GSH. Two
noncanonical c-Myc binding sites (CACATG, E box) are present in the human GCLC
promoter at −559/−554 and −500/−495 and together with ARE4, are responsible for H2O2-
induced GCLC promoter activity [120]. The signaling pathway activated by H2O2 involves
ERK-mediated phosphorylation and activation of c-Myc [120].
Recently, c-AMP-response element binding protein (CREB) was found to be the key
transcription factor rather than Nrf1 or Nrf2, in binding to ARE4 and induction of GCLC
expression in response to anthocyanin (a flavonoid antioxidant) treatment in HepG2 cells
[121]. The induction occurred when Nrf1 or Nrf2 was silenced using siRNA but whether
CREB binds to the ARE4 alone or requires the participation of other transcription factors is
unknown.
In addition to increased gene transcription, DEM and 4-HNE have also been found to
stabilize the GCLC mRNA [102,122]. However, the mechanism of this effect remains
unknown.
4.2 GCLC post-translational regulation
GCL is also regulated post-translationally. GSH synthesis was inhibited by hormone-
mediated activation of various signal transduction pathways [123,124]. These hormones are
secreted under stressful conditions, many of which have associated lower hepatic GSH
levels [10,13]. The fall in hepatic GSH level occurs by both an increase in sinusoidal GSH
efflux [125,126] and an inhibition of GSH synthesis [123]. This may represent the hepatic
stress response by increasing the systemic delivery of GSH and cysteine and channeling
cysteine to synthesis of stress proteins [127]. We showed that GCLC is phosphorylated
directly by activation of protein kinase A (PKA), protein kinase C (PKC) or Ca2+-
calmodulin kinase II (CMK) [128]. Cultured hepatocytes exhibit basal GCLC
phosphorylation, which increased when treated with DBcAMP or phenylephrine, suggesting
GCLC may be under a basal inhibitory tone [128]. Thus, phosphorylation-
dephosphorylation may be an important physiologic regulator of GCL. Since many
pathologic and toxic conditions can lead to activation of CMK and phosphorylation of
GCLC, inhibition of GCL may further contribute to toxicity. Consistent with this notion is
the report that toxic doses of acetaminophen suppressed hepatic GSH synthesis in rats [129].
GCLC can be cleaved by a caspase 3-dependent mechanism from the full-length 73 kDa to a
60 kDa form during apoptosis induced by TGF-β1, TNFα and α-Fas [45,130]. Cleavage of
GCLC occurs at Asp499 within the sequence AVVD499G, which is located upstream of
Cys553 thought to be important for disulfide bond formation with GCLM [35,45].
Theoretically, this would result in decreased GCL activity but this was not observed during
apoptotic cell death [45]. Cleavage of GCLC at Asp499 generates a 13 kDa-C-terminal
fragment with a N-terminal glycine residue that is predicted to be a myristoylation site [45].
While myristoylation was demonstrated when a GCLC fragment was overexpressed [131],
whether this actually occurs during apoptotic cell death remains unclear.
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Recently the lipid peroxidation product 4-HNE was shown to directly adduct GCLC Cys553
(and GCLM Cys35)
in vitro
[132]. Formation of 4-HNE GCLC adduct increased the activity
of monomeric GCLC but inhibited formation of GCL holoenzyme and lowered GCL
holoenzyme activity [132]. In cells where GCLC predominates, this mechanism may allow
increased GSH synthesis by increasing the enzymatic activity of monomeric GCLC.
However, whether this occurs
in vivo
remains to be examined.
4.3 Regulation of GCLM
GCLM plays a critical regulatory role on the overall function of GCL [38,43]. The two
subunits of GCL are often coordinately induced by oxidative stress but as described above,
hormones (insulin, hydrocortisone, TGF-β1) and rapid growth induce GCLC selectively in
the liver. The exception is HGF, which induced the expression of both GCL subunits in
hepatocytes under low-density condition [29]. GCLM is induced by xenobiotics such as β-
NF and TBH [92,93,104,105,133,134]. Similar to the human GCLC, up-regulation of the
human GCLM by β-NF involved binding of transcription factor Nrf2 (possibly in complexes
with other Jun or Maf proteins as in GCLC) to a functional ARE/EpRE site located at −302
of the human GCLM [104,134]. Also, there is a critical c-Myc-binding E-box at
−1609/−1604 of the human GCLM promoter that in conjunction with the proximal ARE,
mediate the full induction of the GCLM promoter under H2O2-induced stress [120]. Nrf2 is
also required for GCLM induction by 15d-PGJ2 and physiological 4-HNE concentrations
[84–85]. Transcription factors and
cis
-acting elements important for mouse and rat GCLM
genes are similar to the human gene. Fibroblast cells derived from Nrf1 and Nrf2 knockout
mice have lower GSH levels and reduced basal expression of GCLM [107,118]. The rat
GCLM promoter also has a functional ARE element (−295 to −285) [97]. This ARE element
is important for basal expression and TNFα-mediated induction of rat GCLM [97]. AP-1,
NFκB and Nrf2 are positive regulators of the rat GCLM gene and are induced by TNFα
treatment [97]. While AP-1 and Nrf2 have direct effects on the rat GCLM promoter, NFκB
activates it indirectly via AP-1 [97]. Not all inducers of oxidative stress induce GCLM.
Ethanol and TGF-β1 treatments do not affect rat GCLM expression [73,135]. The reason for
this discordance is not clear. Finally, GCLM expression also changes in a biphasic manner
during BDL and when hepatocytes are treated with lithocholic acid [90,136 - see below
under cholestatic liver injury].
Post-transcriptional regulation of GCLM also occurs with 4-HNE treatment, which
increased the stability of GCLM mRNA by an unknown mechanism that required
de novo
protein synthesis [122]. This is in contrast to the effect of 4-HNE on GCLC mRNA stability,
which did not require
de novo
protein synthesis. As3+ increased GCLM mRNA stability in
addition to GCLM transcription but the mechanism is unclear [87].
5. Regulation of GSH synthase (GS)
GS has received relatively little attention in the field of GSH biosynthesis. GS is composed
of two identical subunits and is not subject to feedback inhibition by GSH [48]. GS
deficiency in humans can result in dramatic metabolic consequences because the
accumulated γ-glutamylcysteine is converted to 5-oxoproline, which can cause severe
metabolic acidosis, hemolytic anemia and central nervous system damage [137,138]. Choi et
al described decreased hepatic GSH levels, which correlated with reduced GS activity in Tat
transgenic mice [139]. A decrease in GS activity alone without a change in GCL and a fall
in GSH levels occurred after surgical trauma in human skeletal muscle [50]. These findings
seem to contradict the notion that GCL is rate-limiting. Although the specific activity of GS
is normally 2 to 4 times that of GCL activity in normal liver [64,140], this may not be the
case in other tissues and under stressful conditions. In fact, in normal human skeletal
muscle, the specific activity of GS is only 36% higher than that of GCL [50]. Surgical
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trauma selectively reduced GS activity, which probably became rate limiting [50]. Recently,
all-trans retinoic acid (ATRA), was shown to induce the expression of GS selectively (no
effect on GCLC or GCLM) and GSH levels in myeloid-derived suppressor cells [141].
Taken together, these results suggest regulation of GS may also be important in determining
the overall GSH synthetic capacity under certain conditions and especially in non-hepatic
tissues.
We found treatments that increase the expression of both GCL subunits, such as DEM,
buthionine sulfoximine (BSO), TBH, TNFα and HGF treatment of cultured rat hepatocytes
and thioacetamide (TAA) treatment of rats, also increased the expression of GS [26,29,97].
In contrast, treatments that increase the expression of GCLC alone such as insulin,
hydrocortisone in cultured hepatocytes and ethanol feeding
in vivo
, had no influence on GS
expression. There are exceptions, one is liver regeneration after partial hepatectomy, another
is HCC, and a third is liver-specific retinoid X receptor α (RXRα) knockout mice. GS
mRNA levels changed in parallel to that of GCLC in all three conditions while GCLM
mRNA levels were unchanged [26,51,142]. We speculated that when GCL is induced
tremendously, the step catalyzed by GS might become limiting. A coordinated induction in
the activity of both enzymes would further enhance GSH synthesis capacity. Consistent with
this hypothesis, treatments that induced only GCLC increased the GSH synthesis capacity
by 50 to 100% [64,135], whereas treatments that induced both GCLC and GS expression
increased the GSH synthesis capacity by 161–200% [27,140].
Given the coordinated regulation of GCL and GS, it is not surprising that transcriptional
regulation of these genes is quite similar. For the rat GS promoter, AP-1 serves as an
enhancer directly while NF-1 acts as a repressor [143]. NFκB can also activate the rat GS
promoter, albeit indirectly via AP-1 [97]. Both Nrf1 and Nrf2 overexpression induced the
human GS promoter activity [144]. The human GS promoter contains two regions with
homology to the NFE2 (nuclear factor erythroid 2) motif that are required for basal activity
[144]. ATRA, which works via RXR, induced the expression of GS selectively and GSH
levels in myeloid-derived suppressor cells [141]. ATRA treatment for 48 hours also
increased GSH levels in mononuclear cells isolated from patients with metastatic renal cell
carcinoma, but GS expression was not examined [141]. The mechanism of ATRA’s
inductive effect on GS expression required ERK1/2 signaling but the mechanism is not
clear, as ATRA treatment did not influence the expression of Nrf2 or NFκB and it had no
effect on the GS promoter activity [141]. This suggests the possibility of post-transcriptional
regulation of GS by ATRA but this remains to be examined. Post-translational regulation of
GS has not been reported.
6. Dysregulation of GSH synthesis
There is accumulating data that reduced GSH levels occur in many human diseases and they
contribute to worsening of the condition [4]. While oxidative injury plays a dominant role in
GSH depletion in many of these disorders, some are causally related to reduced expression
of GSH synthetic enzymes [13]. In the most severe cases, polymorphisms of GCLC and/or
GCLM that result in significantly reduced GCL expression and activity can present with
severe phenotype including hemolytic anemia, aminoaciduria and spinocerebellar
degeneration [reviewed in 45]. GCLC and GCLM polymorphisms have been reported in
many disorders, including schizophrenia, cardiovascular diseases, stroke, and asthma
[45,145,146]. Outside of polymorphism, decreased GSH synthesis occurs during aging,
diabetes mellitus, fibrotic diseases (including cystic fibrosis and pulmonary fibrosis),
endotoxemia, and several hepatic disorders such as cholestatic and alcoholic liver injury
[13,15]. The opposite situation, namely increased GSH synthesis, plays an important role in
conferring drug and/or radiation resistance to many different cancers [13]. Targeting this
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increase in GCL activity using BSO (the irreversible GCL inhibitor) is now often used as an
adjuvant chemotherapeutic agent in cancer treatment [13]. Given the central role of hepatic
GSH in systemic GSH homeostasis, four liver disorders (cholestasis, endotoxemia, alcohol
and fibrosis) where decreased GSH synthesis may participate in the pathogenesis of liver
injury are described in more detail.
6.1. Cholestasis
Cholestasis is the underlying mechanism for many chronic liver diseases. The underlying
mechanism for cell toxicity is thought to be retention of toxic bile acids, which can cause
oxidative stress, apoptosis, fibrosis leading to cirrhosis [147,148]. Recently we used the
BDL model in mice and showed that hepatic expression of GSH synthetic enzymes
increased early on likely as an adaptive response to oxidative stress but decreased markedly
along with GSH levels during later stages of BDL [136]. A key observation from this study
was the fall in Nrf2 nuclear binding to the ARE two weeks after BDL. A similar pattern of
early induction followed by fall in GSH synthetic enzymes also occurred when Huh-7 cells
(a human hepatoma cell line) was treated with lithocholic acid [90]. In both BDL and
lithocholic acid-treated Huh-7 cells, the fall in expression of GSH synthetic enzymes
coincided with an increase in the expression of several Maf proteins (c-Maf, MafG and
MafK) as well as increased c-Maf and MafG nuclear binding to ARE. MafG and MafK are
small Mafs that have been reported to heterodimerize with Nrf2 to either activate or repress
ARE-dependent genes [108,149]. Small Mafs lack transcriptional activation domain and can
form homodimers to repress ARE-mediated gene expression [150]. In addition, large Maf
protein such as c-Maf can bind to ARE as homodimers and heterodimers with small Mafs
(but not Nrfs) to repress ARE-mediated gene expression [151]. Given these known effects of
small Mafs and c-Maf, we speculated that the induction in Mafs and displacement of Nrf2
from nuclear binding to ARE during cholestasis might have caused the fall in the expression
of GSH synthetic enzymes. Consistent with this, blocking either c-Maf or MafG induction
during BDL protected against the fall in expression of GSH synthetic enzymes, GSH levels
and BDL-induced liver injury [90]. Interestingly, ursodeoxycholic acid (UDCA), the only
medication approved by the FDA for the treatment of primary biliary cirrhosis [152], a
chronic cholestatic disorder, and SAMe were able to raise nuclear Nrf2 level, block the
increase in MafG and c-Maf expression, protect against the fall in expression of GSH
synthetic enzymes and GSH levels in these models [90,136]. Combining UDCA and SAMe
exerted additional benefit, suggesting they have different mechanisms. Murine cholestatic
liver injury is the first example that illustrates the importance of Maf proteins on ARE-
dependent gene expression in liver pathology.
6.2 Endotoxemia
Lipopolysaccharide (LPS, synonymous as endotoxin) is a major constituent of the outer cell
wall of all gram-negative bacteria that can trigger the synthesis and release of pro-
inflammatory cytokines and inducible nitric oxide synthase (iNOS) [153,154]. Liver clears
gut-derived LPS [154]. This explains why endotoxemia occurs in cirrhotic patients and the
degree of endotoxemia correlates with the degree of liver failure [154]. Endotoxemia also
participates in worsening of alcoholic liver disease and non-alcoholic steatohepatitis
[154,155]. Endotoxemia lowers GSH levels in the liver [156,157], peritoneal macrophages
and lymphocytes [158]. Septic patients have lower blood GSH:GSSG ratios [159].
Exogenous GSH treatment suppressed LPS-induced systemic inflammatory response and
reduced mortality [160]. GSH level is an important variable that determines susceptibility to
LPS-induced injury in multiple tissues [157,160,161]. This may be related to GSH’s ability
to influence toll like receptor 4 (TLR4) signaling. Specifically, LPS-induced mortality and
TNFα secretion were higher when GSH level was reduced [162]. The fall in GSH is
multifactorial. In liver, increased GSH efflux and increased oxidative stress both contribute
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[153,156]. One study showed that a major mechanism of hepatic GSH depletion during
endotoxemia is a fall in GCLC mRNA level and GCL activity [157]. Consistent with this,
we found that hepatic GSH level fell more than 50% following LPS, coinciding with a
comparable fall in the mRNA and protein levels of GCLC and GCLM (50–60%) [163]. GS
expression fell to a lesser extent (40% fall). SAMe pretreatment protected against liver
injury and prevented the fall in GCLC and GCLM expression and GSH level. Nearly
maximum inhibition in the expression of GSH synthetic enzymes occurred as early as six
hours after LPS administration [163]. The molecular mechanisms remain to be elucidated.
6.3 Alcohol
Alcoholic liver disease patients have low hepatic and plasma GSH levels due to multiple
mechanisms such as oxidative stress, nutritional deficiency and abnormalities in the
methionine metabolic pathway that impairs cysteine availability [164–166]. In addition, we
found a 50% fall in the mRNA levels of GCLC and GS (GCLM was unchanged) in patients
hospitalized for alcoholic hepatitis [166]. The mechanism for this is unclear but these
abnormalities may contribute to the high morbidity and mortality associated with this
disorder.
6.4 Fibrogenesis
HSCs are the key effectors in hepatic fibrogenesis [167]. HSCs reside in the space of Disse
and in normal liver are the major storage sites of vitamin A. Following chronic liver injury,
HSCs proliferate, lose their vitamin A and undergo a major phenotypical transformation to
α-smooth muscle actin (α-SMA) positive activated HSCs, which produce a wide variety of
collagenous and non-collagenous extracellular matrix (ECM) proteins [168]. The
profibrogenic potential of activated HSCs is due to their capacity to synthesize fibrotic
matrix proteins and components that inhibit fibrosis degradation. Pro-fibrogenic factors
include TGF-β [169], connective tissue growth factor [170], leptin [171] and platelet derived
growth factor [172]. Activation of HSC is mediated by various cytokines and ROS released
from damaged hepatocytes and activated Kupffer cells [173]. Hence, inhibition of HSC
activation and its related events such as ECM formation and cellular proliferation are
important targets for therapeutic intervention. In both hepatic and pulmonary fibrosis, TGF-
β1 has been shown to target GSH synthesis (see above under GCLC regulation) [15,73,82].
EGCG and curcumin, two agents that exert anti-fibrotic effect in hepatic HSCs, require de
novo GSH synthesis to exert this effect [73,82]. In BDL, preventing the fall in hepatic GSH
also resulted in amelioration of hepatic fibrosis [90]. Consistent with the importance of GSH
in hepatic fibrogenesis, we found that a lower hepatic GSH level greatly potentiated BDL-
induced fibrosis and if induction in GCLC expression was blocked (by using RNAi), the
therapeutic efficacy of UDCA and SAMe was nearly lost [174]. We also established that
GCLC expression is a critical factor in determining the phenotype of rat HSCs (GCLM and
GS were unchanged at the protein level) [174]. Specifically, GCLC expression fell during
HSC activation and increased as activated HSCs revert to quiescence. Blocking the increase
in GCLC expression kept HSCs in an activated state. Although activated HSCs have
increased nuclear MafG level, formation of Nrf2/MafG heterodimer and binding to ARE is
greatly diminished. In contrast, quiescent HSCs have markedly lower total nuclear MafG
level but increased Nrf2/MafG heterodimerization and binding to ARE. This is due to
enhanced sumoylation of Nrf2 and MafG by SUMO-1 in the quiescent state, which
facilitated heterodimerization and binding to ARE [174]. Thus, a key mechanism that
controls Nrf2/MafG trans-activation of GCLC ARE is sumoylation by SUMO-1 in HSCs.
Taken together, a fall in GSH facilitates activation of HSCs and fibrosis to proceed.
Targeting this is an attractive therapeutic strategy that yielded promising results in animal
models of pulmonary and hepatic fibrosis [15, 90] but human trials have not been as positive
[15].
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7. Concluding remarks
Up until recently, most of the literature on GSH synthesis has focused on understanding how
the enzymes are regulated transcriptionally and post-transcriptionally. There are now
increasing evidence that show dysregulation of GSH synthesis in multiple conditions, such
as aging, diabetes, pulmonary and hepatic fibrosis, alcoholic and cholestatic liver injuries.
Some of these have been confirmed to occur also in humans. GCLC and GCLM
polymorphisms have also gained attention as another determinant of chronic oxidative injury
to various organs. While the status of screening for these polymorphisms remains to be
established, uncovering the molecular mechanisms responsible for the dysregulation in GSH
synthesis may provide novel therapeutic approaches.
Acknowledgments
This work was supported by NIH grant R01DK092407
Abbreviations (in alphabetical order)
4-HNE 4-hydroxynonenal
15d-PGJ215-deoxy-Δ12,14-prostaglandin J2
AP-1 activator protein-1
As3+ trivalent arsenite
α-SMA a-smooth muscle actin
ARE antioxidant response element
ATRA all-trans retinoic acid
BDL bile duct ligation
BHMT betaine homocysteine methyltransferase
β-NF β-naphthoflavone
BSO buthionine sulfoximine
CBS cystathionine β synthase
CMK Ca2+-calmodulin kinase II
CNC-bZIP cap ‘n’ collar-basic leucine zipper proteins
CREB c-AMP-response element binding protein
DEM diethyl maleate
ECM extracellular matrix
EpRE electrophile response element
GCL glutamate-cysteine ligase
GCLC GCL-catalytic subunit
GCLM GCL-modifier subunit
GGT γ-glutamyltranspeptidase
GPx GSH peroxidase
GS GSH synthase
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GSH glutathione
GSSG oxidized GSH
HCC hepatocellular carcinoma
Hcy homocysteine
HGF hepatocyte growth factor
HSC hepatic stellate cell
iNOS inducible nitric oxide synthase
LPS lipopolysaccharide
MAT methionine adenosyltransferase
MRE metal response element
MS methionine synthase
MT methyltransferase
NFE2 nuclear factor erythroid 2
NO nitric oxide
Nrf2 nuclear factor-erythroid 2 related factor 2
PKA protein kinase A
PKC protein kinase C
RNS reactive nitrogen species
RXRαretinoid X receptor α
ROS reactive oxygen species
SAH S-adenosylhomocysteine
SAMe S-adenosylmethionine
TAA thioacetamide
TBH tert-butyl hydroquinone
TGF-β1transforming growth factor-β1
TLR4 toll like receptor 4
TNFαtumor necrosis factor α
UDCA ursodeoxycholic acid
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Highlights
•GSH regulates antioxidant defense, growth, death, immune function, and
fibrogenesis.
•GSH is synthesized via two enzymatic steps that are regulated at multiple levels.
•GSH synthesis is dysregulated in multiple human diseases.
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Fig. 1. GSH synthesis
Synthesis of GSH occurs via a two-step ATP-requiring enzymatic process. The first step is
catalyzed by glutamate-cysteine ligase (GCL), which is composed of catalytic and modifier
subunits (GCLC and GCLM). This step conjugates cysteine with glutamate, generating γ-
glutamylcysteine. The second step is catalyzed by GSH synthase, which adds glycine to γ-
glutamylcysteine to form γ-glutamylcysteinylglycine or GSH. GSH exerts a negative
feedback inhibition on GCL.
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Fig. 2. Antioxidant function of GSH
Aerobic metabolism generates hydrogen peroxide (H2O2), which can be metabolized by
GSH peroxidase (GPx) in the cytosol and mitochondria, and by catalase in the peroxisome.
GSSG can be reduced back to GSH by GSSG reductase (GR) at the expense of NADPH,
thereby forming a redox cycle. Organic peroxides (ROOH) can be reduced by either GPx or
GSH S-transferase (GST). GSH also plays a key role in protein redox signaling. During
oxidative stress, protein cysteine residues can be oxidized to sulfenic acid (Prot-SOH),
which can react with GSH to form protein mixed disulfides Prot-SSG (glutathionylation),
which in turn can be reduced back to Prot-SH via glutaredoxin (Grx) or sulfiredoxin (Srx).
This is a mechanism to protect sensitive protein thiols from irreversible oxidation and may
also serve to prevent loss of GSH under oxidative conditions. The ability of the cell to
reduce GSSG to GSH may be overcome during severe oxidative injury, leading to an
accumulation of GSSG. To prevent a shift in the redox equilibrium, GSSG can either be
actively transported out of the cell or react with a protein sulfhydryl (Prot-SH) to form a
mixed disulfide (Prot-SSG).
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Fig. 3. GSH is a continuous source of cysteine via the γ-glutamyl cycle
GSH is transported out of the cell where the ecto-enzyme γ-glutamylpeptidase (GGT)
transfers the γ-glutamyl moiety of GSH to an amino acid (the best acceptor being cystine),
forming γ-glutamyl amino acid and cysteinylglycine. The γ-glutamyl amino acid can then
be transported back into the cell and once inside, the γ-glutamyl amino acid can be further
metabolized to release the amino acid and 5-oxoproline, which can be converted to
glutamate and reincorporated into GSH. Cysteinylglycine is broken down by dipeptidase
(DP) to generate cysteine and glycine, which are also transported back into the cell to be
reincorporated into GSH. Most of the cysteine taken up is incorporated into GSH while the
rest is incorporated into newly synthesized proteins and/or broken down into sulfate and
taurine.
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Fig. 4. Hepatic methionine metabolism
Liver plays a central role in methionine catabolism as up to half of the daily intake of
methionine is catabolized to S-adenosylmethionine (SAMe) in the liver in a reaction
catalyzed by methionine adenosyltransferase (MAT). SAMe is the principal biological
methyl donor and donates its methyl group to a large variety of acceptor molecules in
reactions catalyzed by methyltransferases (MTs). S-adenosylhomocysteine (SAH),
generated as a result of transmethylation, is a potent inhibitor of all transmethylation
reactions. To prevent SAH accumulation, it is hydrolyzed to homocysteine and adenosine is
through a reversible reaction catalyzed by SAH hydrolase, whose thermodynamics favors
biosynthesis rather than hydrolysis. Prompt removal of homocysteine and adenosine ensures
SAH is hydrolyzed. Homcysteine can be remethylated to form methionine via methionine
synthase (MS), which requires folate and vitamin B12 and betaine homocysteine
methyltransferase (BHMT), which requires betaine. In hepatocytes, homocysteine can also
undergo conversion to cysteine (Cys) via the transsulfuration pathway, a two-step enzymatic
process catalyzed by cystathionine β-synthase (CBS) and cystathionase, both requiring
vitamin B6. Liver has the highest activity of transsulfuration, which allows methionine and
SAMe to be effectively utilized as GSH precursor.
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