Download - GLUTATHIONE TRANSFERASES
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GLUTATHIONE TRANSFERASES
Ralf MorgensternInstitute of Environmental MedicineKarolinska Institutet
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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
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THREE SUPERFAMILIES
• SOLUBLE GLUTATHIONE-TRANSFERASES (25 kDa, dimers) aerobic organisms
• MEMBRANE BOUND GLUTATHIONE- TRANSFERASEs (17 kDa, trimer) aerobic organisms
• FOSFOMYCIN RESISTANCE PROTEIN (Fos A) (16 kDa, dimer) bacterial
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FOSFOMYCIN RESISTANCE (Fos A)
• Bacterial (plasmid or chromosomal)
• Specific
• Fosfomycin is a stable! epoxide that inhibits cell wall-synthesis in bacteria
Fosfomycin (antibiotic)
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CYTOSOLIC GLUTATHIONE TRANSFERASES
• SEVERAL FAMILIES: alfa, mu, pi, theta, sigma, zeta, omega, beta, phi (incl. ≥1)
Form dimers:Within a familyhomo- and heterodimers
Monomers:
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Evolutionary aspects
Thioredoxinfold
Domainaddition
Domaininsertion
GST Theta
MitochondrialGST Kappa
Cytosolic GSTs
Alpha, Mu, Pi,Sigma, Beta Zeta, Omega, Phi, Tau,Delta, etc
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Human soluble GSTs
Gene
family
alpha mu theta pi zeta sigma kappa omega
Genes A1-A5
M1-M5
T1,T2 P1 Z1 S1 K1 O1
Chromo-some
6p 1p 22q 11q 14q 4q 7q 10q
Enzyme Nomenclature: GSTP1-1 or GSTA1-2.
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Tissue-distribution (human)
1, Standard2, brain3, heart4, kidney5, liver6, lung7, pancreas8, prostate9, muscle10, intestine11, spleen12, testis
Sherratt et al., Biochem. J. (1997) 326, 837
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DIMER-STRUCTURE
G-site
H-site
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GSH binding
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Making GSH more reactive
GSH GS- +H+
OH
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)
Arg+
pKaGSH lowered from 9
to ≈ 6 in the enzyme
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GSH is bound in anExtended Conformationwhere all possibleinteractions are used
Tyr-OH
GS- thiolate
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An model second substrate and convenient assay
HCl
Cl
NO2
GS
GSH + +
NO2
NO2
NO2
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New fluorogenic substrates
GST
Ålander et al, Anal. Biochem. (2009) 390, 52.
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The H-site
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Multiple Functions
BCNU (cytostatic)
Aflatoxin (carcinogen)
Atrazine (herbicide)
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Reactive compounds are common in biology
• Cyanobacteria: microcystine
GSH
• Mustard oil: allylisothiocyanate
N
C
S
N
C
S
S
G
N
N
N
N
N
X
N
N
Z
O
O
C
O
O
H
O
C
H
2
O
O
O
O
O
C
O
O
H
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Reactive compounds are formed continuously in the cell
Lipid peroxidation gives rise to:
Hydroxyalkenals:
Hydroperoxides:
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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
-L-Glu Gly
-L-Glu-L-Cys-Gly L-Cys-Gly L-Cys N-Ac-L-Cys
N-Acetylation
SX SXSXSX
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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
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Genetic variation in human glutathione transferase Mu
English 45%Japanese 48%Indian 35%Micronesia 100%Chinese 58%French 43%Scots 62%
% of population that are homozygous deleted for the gene.Persons that lack the gene are more susceptible to certain forms of cancer.
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Drug resistance
BCNU (cytostatic drug)
Up-regulation of GST seen in many tumours could contribute to resistance
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GST protection
Yang et al JBC276, 19220
H2O2 is not a substrate for GTSs
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GSTP knockout leads to
increased c-Jun signalling
= increased proliferation
GSTP
GSTPGSTP
GSTPStress (H2O2)
GSTP
JNK JNK
C-JunP
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GSTP catalyses protein S-glutathionylation (H2O2 challenge)
Townsend et al JBC 284, 436
Tyr 7, and Cys 47/101
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GSTP & Prdx6 = GSH Peroxidase
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Regulation by Induction
GLUTATHIONE-TRANSFERASE-ACTIVITYin butterfly larvae
depends on dietand treatment withchemicals: e.g.Endosulfan (insecticide)
Willovleaves
Apple leaves
+ endosulfan
+ endosulfan
C
l
C
l
C
l
C
l
O
S
O
O
C
Cl2
DIET
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Chemoprevention depends on Nrf2 regulation
Nrf2
Reactive compounds
Nrf2
Antioxidant Response Element
-SH
Cytosol
GSTsQuinone reductaseGSH synthesis
S
O
N C SSulphoraphane
Glukosinolat
nuclei
Keap
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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)
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GSTP, Cytosolic and more
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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
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The MAPEG theme: reactive lipid
Oxygenated arachidonic acid
Prostaglandin ELeukotrienes
Peroxidized lipids
Detoxification by Microsomal Glutathione Transferases (MGSTs) 1-3
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MGST1TRIMER3-D model
(3.2 Å)
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Peroxidized lipid substrates
As GlutathionePeroxidases (GPX)
Conjugation ofreactive lipidperoxidationproducts
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cGST/GPX1
PHGPX4
MGST1
Location, location, location....
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MTT
0 25 50 75 100 125 150 175 2000
102030405060708090
100110120
Sense
AS
MCF7wt
[HNE] (µM)
Viability (%)
Cellular protection by MGST1
MGST1Trans-fected
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MGST1 knockout flies display shorter life span
Knock-outs Toba & Aigiki, Gene, 253, 179 (2000)
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SPECIFIC FUNCTIONS
Stimuli
AA
FLAP*5-LO
LTA4
LTC4S*
LTC4Airway-tonus(Asthma)
AA
PGH2
COX
PGES*
PGE2
FeverPainInflam-mation*MAPEG members
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PGE synthase
PGH2
PGE2
O
O
O
H
O
O
O
O
O
Requires GSH
OH
OH
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MGST1 MGST2 MGST3LeukotrieneC4 synthase
5-Lipoxy-genase
activatingprotein
PGES
Glutathione peroxidases
Tissue distribution:WIDE NARROW
GST:s
NARROW
GSH-dep.oxido-reductase
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MGST1 activation
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MGST1 is activatedby sulfhydryl reagents
SHSHSH
+N
OO
NEM
SNEMSNEMSNEM
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!
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Thiolate anion formation is activated
Activation increasesthe rate of thiolateanion formation(not the chemical step)
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Activation of MGST1 by reactive intermediates in vivo (2-3 fold)
O
O
O
O
Diethylmaleate (direct)
CCl4 • CCl3P450
Br
Br
Br
G
S
G
S
GST
+
Thiiranium ion
+ Br-
+ Br-
GSH
SpontaneousAcetaminophen
Reactive quinoneimine
O
H
O
P450
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Activation Mechanisms of MGST1
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Activation of MGST1 by S-thiolationIn vitro by GSSG
In vivo by hydroperoxide
GSSG/GSH ratio = 50 at half maximal activation
Sies et al, ABB 322, 288
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Capacity and throughputCAPACITY: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
CONCLUSIONGlutathione dependent protection has to be highly abundant and efficient to serve as an interception system
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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.
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Examples of drugs that are conjugated to GSH
• Paracetamol (analgesic, antipyretic)
• Carbamazepine (analgesic)
• Indomethacin (anti-inflammary)
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Paracetamol
N
O
O
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Indomethacin
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The nucleophile substrate
Most Aerobic Organisms contain:
• mM concentrations of the low molecular weight nucleophile,
Glutathione, -L-Glu-L-Cys-Gly (GSH)