GLUTATHIONE
TRANSFERASES
Ralf Morgenstern
Institute of Environmental
Medicine
Karolinska Institutet
Current themes
•
•
•
•
•
•
•
GSTs and intracellular signalling pathways
MAPEG and eicosanoid signalling
Redox regulation (Protein S-glutathionylation)
Oxidative stress protection
Drug resistance in tumors
Chemo-prevention
Tools for bioengineering
THREE SUPERFAMILIES
• SOLUBLE GLUTATHIONE-TRANSFERASES
(25 kDa, dimers) aerobic organisms
• MEMBRANE BOUND GLUTATHIONETRANSFERASEs (17 kDa, trimer) aerobic organisms
• FOSFOMYCIN RESISTANCE PROTEIN (Fos A)
(16 kDa, dimer) bacterial
FOSFOMYCIN RESISTANCE
(Fos A)
Fosfomycin (antibiotic)
• Bacterial (plasmid or chromosomal)
• Specific
• Fosfomycin is a stable! epoxide that
inhibits cell wall-synthesis in bacteria
CYTOSOLIC GLUTATHIONE
TRANSFERASES
• SEVERAL FAMILIES: alfa, mu, pi, theta,
sigma, zeta, omega, beta, phi (incl. ≥1)
Monomers:
Form dimers:
Within a family
homo- and
heterodimers
Evolutionary aspects
Domain
addition
Thioredoxin
fold
Domain
insertion
GST Theta
Mitochondrial
GST Kappa
Cytosolic GSTs
Alpha, Mu, Pi,
Sigma, Beta Zeta,
Omega, Phi, Tau,
Delta, etc
Human soluble GSTs
Gene
family
alpha
mu
theta
Genes
A1A5
M1M5
1p
Chromo- 6p
some
pi
zeta
sigma
kappa
omega
T1,T2 P1
Z1
S1
K1
O1
22q
14q
4q
7q
10q
11q
Enzyme Nomenclature: GSTP1-1 or GSTA1-2.
Tissue-distribution (human)
1, Standard
2, brain
3, heart
4, kidney
5, liver
6, lung
7, pancreas
8, prostate
9, muscle
10, intestine
11, spleen
12, testis
Sherratt et al., Biochem. J. (1997) 326, 837
DIMER-STRUCTURE
H-site
G-site
GSH binding
Making GSH more reactive
GSH
GSOH
pKaGSH
lowered from 9
to ≈ 6 in the enzyme
+H+
Arg+
Tyrosine
or Serine
(backbone amide?)
GS- thiolate is 109 times more reactive than the protonated thiol
(Thiolate/CDNB ≈ 5 M-1 s-1; Selenolate/CDNB ≈ 23 M-1 s-1)
GSH is bound in an
Extended Conformation
where all possible
interactions are used
GS- thiolate
Tyr-OH
An model second substrate and
convenient assay
Cl
GS
NO2
NO2
GSH
+
+
NO2
NO2
HCl
New fluorogenic substrates
GST
Ålander et al, Anal. Biochem. (2009) 390, 52.
The H-site
Multiple Functions
Aflatoxin (carcinogen)
BCNU (cytostatic)
Atrazine (herbicide)
Reactive compounds are
common in biology
• Cyanobacteria:
microcystine
O
COOH
N
N
O
O
O
N
CH 2
O
N
O
N
Z
N
N
O
• Mustard oil:
allylisothiocyanate
N
C
X
COOHO
S
GSH
N
C
S
SG
Reactive compounds are formed
continuously in the cell
Lipid peroxidation
gives rise to:
Hydroxyalkenals:
Hydroperoxides:
Conjugate export and processing
• GSH conjugates are exported out of the cell by
membrane transporters called MDR (multidrug
resistance proteins)
• Conjugates are often processed to mercapturic
acids before excretion in urine or bile
N-Acetylation
g-L-Glu-L-Cys-Gly
SX
L-Cys-Gly
g-L-Glu
SX
L-Cys
Gly
SX
N-Ac-L-Cys
SX
Knock-outs
• GSTP null mice are more susceptible to
skin and lung cancer
• GSTA4 null mice are more susceptible to
bacterial infection and oxidative stress
• GSTBeta null bacteria are more susceptible
to oxidative stress
Genetic variation in human
glutathione transferase Mu
English
Japanese
Indian
Micronesia
Chinese
French
Scots
45%
48%
35%
100%
58%
43%
62%
% of population that are homozygous deleted for the gene.
Persons that lack the gene are more susceptible to certain forms
of cancer.
Drug resistance
BCNU (cytostatic drug)
Up-regulation of GST seen in many tumours could contribute
to resistance
GST protection
H2O2 is not a
substrate for GTSs
Yang et al JBC276, 19220
GSTP
knockout
leads to
increased cJun signalling
= increased
proliferation
Stress
(H2O2)
GSTP
GSTP
GSTP
GSTP
P
C-Jun
GSTP
JNK
JNK
GSTP catalyses protein Sglutathionylation (H2O2 challenge)
Tyr 7, and Cys 47/101
Townsend et al JBC 284, 436
GSTP & Prdx6 = GSH Peroxidase
Regulation by Induction
GLUTATHIONETRANSFERASEACTIVITY
in butterfly larvae
+ endosulfan
Cl
O
Cl
CCl 2
Cl
S
O
Cl
+ endosulfan
depends on diet
Willov
Apple leaves
and treatment with
leaves
chemicals: e.g.
DIET
Endosulfan (insecticide)
O
Chemoprevention depends on
Nrf2 regulation
Reactive compounds
Keap
-SH Nrf2
GSTs
Quinone reductase
GSH synthesis
Cytosol
Glukosinolat
O
S
N C
Sulphoraphane
Nrf2
S
Antioxidant
Response Element
nuclei
Multiple subcellular distribution
• MGST1: Endoplasmic reticulum,
outer mitochondrial membrane and
plasma membrane
• Soluble GSTs: Cytosol,
mitochondria, nucleus and some
forms show affinity for (plasma)
membrane(s)
GSTP, Cytosolic and more
The MAPEG superfamily
• MAPEG = Membrane Associated Proteins
in Eicosanoid and Glutathione metabolism
• Membrane bound glutathione transferases
• Prostaglandin E2 synthase
• Leukotriene C4 synthase
• 5-Lipoxygenase activating protein
The MAPEG theme: reactive
lipid
Oxygenated
arachidonic acid
Prostaglandin E
Leukotrienes
Peroxidized lipids
Detoxification by
Microsomal Glutathione
Transferases (MGSTs) 1-3
MGST1
TRIMER
3-D model
(3.2 Å)
Peroxidized lipid substrates
Conjugation of
reactive lipid
peroxidation
products
As Glutathione
Peroxidases
(GPX)
Location, location, location....
cGST/GPX1
MGST1
PHGPX4
Cellular protection by MGST1
Viability (%)
MTT
120
110
100
90
80
70
60
50
40
30
20
10
0
Sense
AS
MCF7wt
MGST1
Transfected
0
25
50
75
100 125 150 175 200
[HNE] (µM)
MGST1 knockout flies display
shorter life span
Knock-outs
Toba & Aigiki, Gene, 253, 179 (2000)
SPECIFIC FUNCTIONS
Airwaytonus
(Asthma)
LTC4
LTA4
LTC4S*
AA 5-LO
FLAP*
PGE2
Stimuli
PGH2 PGES*
*MAPEG members
AA
COX
Fever
Pain
Inflammation
PGE synthase
O
O
O
O
PGH2
OH
Requires GSH
O
O
O
HO
OH
PGE2
Tissue distribution:
NARROW
PGES
MGST1
WIDE
NARROW
MGST2 MGST3
5-LipoxyLeukotriene genase
C4 synthase activating
protein
GSH-dep.
oxidoreductase
GST:s
Glutathione peroxidases
MGST1 activation
MGST1 is activated
by sulfhydryl reagents
SH SH
SH
SNEM SNEMSNEM
N
+
O
O
NEM
2 µmol/min mg
30 µmol/min mg
At the single cysteine-49 of the homo-trimer (subunits Mr ≈ 17 kDa)
Activation does occur under toxic and oxidative stress in vivo!
Thiolate anion formation is
activated
Activation increases
the rate of thiolate
anion formation
(not the chemical step)
Activation of MGST1 by reactive
intermediates in vivo (2-3 fold)
OH
Br
O
O
Br
O
GSH GST
O
O
Br
Diethylmaleate (direct)
CCl4 P450 • CCl3
+ Br-
GS
Acetaminophen
P450
Reactive
quinoneimine
Spontaneous
GS
+
Thiiranium ion
+ Br-
Activation Mechanisms of
MGST1
Activation of MGST1 by S-thiolation
In vitro by GSSG
GSSG/GSH
ratio = 50 at
half maximal
activation
In vivo by hydroperoxide
Sies et al, ABB 322, 288
Capacity and throughput
CAPACITY:
0.2 mM Glutathione transferase in liver + 5 mM GSH =
25 turnovers empties the liver of GSH (e.g. paracetamol
overdose) Theoretically this can happen in less than a
second!!!!
THROUGHPUT:
Humans excrete 0.1 mmol glutathione
conjugates per day = Equal to one
turnover per enzyme every second day
CON CLUSION
Glutathione dependent protection has
to be highly abundant and efficient
to serve as an interception system
Glutathione transferases
• Highly abundant and diverse protection from
reactive electrophiles
• New functions in cell signalling and redox
processes
• Dynamic regulation
• Defined chemical transformations of important
endogenous mediators and metabolites
• Relevance to inflammation, drug development,
drug resistance, anti-carcinogenesis, antibiotic
resistance and agriculture.
Examples of drugs that are
conjugated to GSH
• Paracetamol (analgesic,
antipyretic)
• Carbamazepine (analgesic)
• Indomethacin (anti-inflammary)
Paracetamol
O
N
O
Indomethacin
The nucleophile substrate
Most Aerobic Organisms contain:
• mM concentrations of the low molecular weight nucleophile,
Glutathione, g-L-Glu-L-Cys-Gly (GSH)