GLUTATHIONE TRANSFERASES Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet.
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Transcript of 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 GLUTATHIONE- TRANSFERASEs (17 kDa, trimer) aerobic organisms
• FOSFOMYCIN RESISTANCE PROTEIN (Fos A) (16 kDa, dimer) bacterial
FOSFOMYCIN RESISTANCE (Fos A)
• Bacterial (plasmid or chromosomal)
• Specific
• Fosfomycin is a stable! epoxide that inhibits cell wall-synthesis in bacteria
Fosfomycin (antibiotic)
CYTOSOLIC GLUTATHIONE TRANSFERASES
• SEVERAL FAMILIES: alfa, mu, pi, theta, sigma, zeta, omega, beta, phi (incl. ≥1)
Form dimers:Within a familyhomo- and heterodimers
Monomers:
Evolutionary aspects
Thioredoxinfold
Domainaddition
Domaininsertion
GST Theta
MitochondrialGST Kappa
Cytosolic GSTs
Alpha, Mu, Pi,Sigma, Beta Zeta, Omega, Phi, Tau,Delta, etc
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.
Tissue-distribution (human)
1, Standard2, brain3, heart4, kidney5, liver6, lung7, pancreas8, prostate9, muscle10, intestine11, spleen12, testis
Sherratt et al., Biochem. J. (1997) 326, 837
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
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
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
-L-Glu Gly
-L-Glu-L-Cys-Gly L-Cys-Gly L-Cys N-Ac-L-Cys
N-Acetylation
SX SXSXSX
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 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.
Drug resistance
BCNU (cytostatic drug)
Up-regulation of GST seen in many tumours could contribute to resistance
GSTP knockout leads to
increased c-Jun signalling
= increased proliferation
GSTP
GSTPGSTP
GSTPStress (H2O2)
GSTP
JNK JNK
C-JunP
GSTP catalyses protein S-glutathionylation (H2O2 challenge)
Townsend et al JBC 284, 436
Tyr 7, and Cys 47/101
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
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
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)
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 ELeukotrienes
Peroxidized lipids
Detoxification by Microsomal Glutathione Transferases (MGSTs) 1-3
Peroxidized lipid substrates
As GlutathionePeroxidases (GPX)
Conjugation ofreactive lipidperoxidationproducts
MTT
0 25 50 75 100 125 150 175 2000
102030405060708090
100110120
Sense
AS
MCF7wt
[HNE] (µM)
Viability (%)
Cellular protection by MGST1
MGST1Trans-fected
SPECIFIC FUNCTIONS
Stimuli
AA
FLAP*5-LO
LTA4
LTC4S*
LTC4Airway-tonus(Asthma)
AA
PGH2
COX
PGES*
PGE2
FeverPainInflam-mation*MAPEG members
MGST1 MGST2 MGST3LeukotrieneC4 synthase
5-Lipoxy-genase
activatingprotein
PGES
Glutathione peroxidases
Tissue distribution:WIDE NARROW
GST:s
NARROW
GSH-dep.oxido-reductase
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!
Thiolate anion formation is activated
Activation increasesthe rate of thiolateanion formation(not the chemical step)
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
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
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
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)