GLUTATHIONE TRANSFERASES

Ralf MorgensternInstitute of Environmental MedicineKarolinska 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

DIMER-STRUCTURE

G-site

H-site

GSH binding

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

GSH is bound in anExtended Conformationwhere all possibleinteractions are used

Tyr-OH

GS- thiolate

An model second substrate and convenient assay

HCl

Cl

NO2

GS

GSH + +

NO2

NO2

NO2

New fluorogenic substrates

GST

Ålander et al, Anal. Biochem. (2009) 390, 52.

The H-site

Multiple Functions

BCNU (cytostatic)

Aflatoxin (carcinogen)

Atrazine (herbicide)

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

GST protection

Yang et al JBC276, 19220

H2O2 is not a substrate for GTSs

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

GSTP & Prdx6 = GSH Peroxidase

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)

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 ELeukotrienes

Peroxidized lipids

Detoxification by Microsomal Glutathione Transferases (MGSTs) 1-3

MGST1TRIMER3-D model

(3.2 Å)

Peroxidized lipid substrates

As GlutathionePeroxidases (GPX)

Conjugation ofreactive lipidperoxidationproducts

cGST/GPX1

PHGPX4

MGST1

Location, location, location....

MTT

0 25 50 75 100 125 150 175 2000

102030405060708090

100110120

Sense

AS

MCF7wt

[HNE] (µM)

Viability (%)

Cellular protection by MGST1

MGST1Trans-fected

MGST1 knockout flies display shorter life span

Knock-outs Toba & Aigiki, Gene, 253, 179 (2000)

SPECIFIC FUNCTIONS

Stimuli

AA

FLAP*5-LO

LTA4

LTC4S*

LTC4Airway-tonus(Asthma)

AA

PGH2

COX

PGES*

PGE2

FeverPainInflam-mation*MAPEG members

PGE synthase

PGH2

PGE2

O

O

O

H

O

O

O

O

O

Requires GSH

OH

OH

MGST1 MGST2 MGST3LeukotrieneC4 synthase

5-Lipoxy-genase

activatingprotein

PGES

Glutathione peroxidases

Tissue distribution:WIDE NARROW

GST:s

NARROW

GSH-dep.oxido-reductase

MGST1 activation

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 Mechanisms of MGST1

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)

Paracetamol

N

O

O

Indomethacin

The nucleophile substrate

Most Aerobic Organisms contain:

• mM concentrations of the low molecular weight nucleophile,

Glutathione, -L-Glu-L-Cys-Gly (GSH)