GLUTATHIONE TRANSFERASES Ralf Morgenstern Institute of Environmental Medicine

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GLUTATHIONE TRANSFERASES Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet

GLUTATHIONE TRANSFERASES Ralf Morgenstern Institute of Environmental Medicine Karolinska Institutet

Current themes • • GSTs and intracellular signalling pathways MAPEG and eicosanoid signalling Redox

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 k. Da, dimers) aerobic organisms • MEMBRANE BOUND

THREE SUPERFAMILIES • SOLUBLE GLUTATHIONE-TRANSFERASES (25 k. Da, dimers) aerobic organisms • MEMBRANE BOUND GLUTATHIONETRANSFERASEs (17 k. Da, trimer) aerobic organisms • FOSFOMYCIN RESISTANCE PROTEIN (Fos A) (16 k. Da, dimer) bacterial

FOSFOMYCIN RESISTANCE (Fos A) Fosfomycin (antibiotic) • Bacterial (plasmid or chromosomal) • Specific •

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,

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

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 Genes A 1 -A 5 M 1 M

Human soluble GSTs Gene family alpha Genes A 1 -A 5 M 1 M 5 Chromo- 6 p some mu 1 p theta pi zeta sigma kappa omega T 1, T 2 P 1 Z 1 S 1 K 1 O 1 22 q 11 q 14 q 4 q 7 q 10 q Enzyme Nomenclature: GSTP 1 -1 or GSTA 1 -2.

Tissue-distribution (human) 1, Standard 2, brain 3, heart 4, kidney 5, liver 6, lung

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

DIMER-STRUCTURE H-site G-site

GSH binding

GSH binding

Making GSH more reactive GSH GSOH p. Ka lowered from 9 to ≈ 6

Making GSH more reactive GSH GSOH p. Ka lowered from 9 to ≈ 6 in the enzyme GSH +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-

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 NO 2 GSH + +

An model second substrate and convenient assay Cl GS NO 2 GSH + + NO 2 HCl

New fluorogenic substrates GST Ålander et al, Anal. Biochem. (2009) 390, 52.

New fluorogenic substrates GST Ålander et al, Anal. Biochem. (2009) 390, 52.

The H-site

The H-site

Multiple Functions Aflatoxin (carcinogen) BCNU (cytostatic) Atrazine (herbicide)

Multiple Functions Aflatoxin (carcinogen) BCNU (cytostatic) Atrazine (herbicide)

Reactive compounds are common in biology • Cyanobacteria: microcystine • Mustard oil: allylisothiocyanate GSH

Reactive compounds are common in biology • Cyanobacteria: microcystine • Mustard oil: allylisothiocyanate GSH

Reactive compounds are formed continuously in the cell Lipid peroxidation gives rise to: Hydroxyalkenals:

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

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 •

Knock-outs • GSTP null mice are more susceptible to skin and lung cancer • GSTA 4 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

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

Drug resistance BCNU (cytostatic drug) Up-regulation of GST seen in many tumours could contribute to resistance

GST protection H 2 O 2 is not a substrate for GTSs Yang et

GST protection H 2 O 2 is not a substrate for GTSs Yang et al JBC 276, 19220

Stress (H 2 O 2) GSTP knockout leads to increased c. Jun signalling =

Stress (H 2 O 2) GSTP knockout leads to increased c. Jun signalling = increased proliferation GSTP P C-Jun GSTP JNK

GSTP catalyses protein Sglutathionylation (H 2 O 2 challenge) Tyr 7, and Cys 47/101

GSTP catalyses protein Sglutathionylation (H 2 O 2 challenge) Tyr 7, and Cys 47/101 Townsend et al JBC 284, 436

GSTP & Prdx 6 = GSH Peroxidase

GSTP & Prdx 6 = GSH Peroxidase

Regulation by Induction GLUTATHIONETRANSFERASEACTIVITY in butterfly larvae + endosulfan depends on diet Willov Apple

Regulation by Induction GLUTATHIONETRANSFERASEACTIVITY in butterfly larvae + endosulfan depends on diet Willov Apple leaves and treatment with leaves chemicals: e. g. DIET Endosulfan (insecticide)

Chemoprevention depends on Nrf 2 regulation Reactive compounds Keap -SH Nrf 2 GSTs Quinone

Chemoprevention depends on Nrf 2 regulation Reactive compounds Keap -SH Nrf 2 GSTs Quinone reductase GSH synthesis Cytosol Glukosinolat O S N C Sulphoraphane Nrf 2 S Antioxidant Response Element nuclei

Multiple subcellular distribution • MGST 1: Endoplasmic reticulum, outer mitochondrial membrane and plasma membrane

Multiple subcellular distribution • MGST 1: 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

GSTP, Cytosolic and more

The MAPEG superfamily • MAPEG = Membrane Associated Proteins in Eicosanoid and Glutathione metabolism

The MAPEG superfamily • MAPEG = Membrane Associated Proteins in Eicosanoid and Glutathione metabolism • Membrane bound glutathione transferases • Prostaglandin E 2 synthase • Leukotriene C 4 synthase • 5 -Lipoxygenase activating protein

The MAPEG theme: reactive lipid Oxygenated arachidonic acid Prostaglandin E Leukotrienes Peroxidized lipids Detoxification

The MAPEG theme: reactive lipid Oxygenated arachidonic acid Prostaglandin E Leukotrienes Peroxidized lipids Detoxification by Microsomal Glutathione Transferases (MGSTs) 1 -3

MGST 1 TRIMER 3 -D model (3. 2 Å)

MGST 1 TRIMER 3 -D model (3. 2 Å)

Peroxidized lipid substrates Conjugation of reactive lipid peroxidation products As Glutathione Peroxidases (GPX)

Peroxidized lipid substrates Conjugation of reactive lipid peroxidation products As Glutathione Peroxidases (GPX)

Location, location. . c. GST/GPX 1 MGST 1 PHGPX 4

Location, location. . c. GST/GPX 1 MGST 1 PHGPX 4

Cellular protection by MGST 1 Transfected

Cellular protection by MGST 1 Transfected

MGST 1 knockout flies display shorter life span Knock-outs Toba & Aigiki, Gene, 253,

MGST 1 knockout flies display shorter life span Knock-outs Toba & Aigiki, Gene, 253, 179 (2000)

SPECIFIC FUNCTIONS Airwaytonus (Asthma) LTC 4 Stimuli LTA 4 LTC 4 S* AA 5

SPECIFIC FUNCTIONS Airwaytonus (Asthma) LTC 4 Stimuli LTA 4 LTC 4 S* AA 5 -LO FLAP* *MAPEG members PGE 2 PGH 2 PGES* AA COX Fever Pain Inflammation

PGE synthase PGH 2 OH Requires GSH PGE 2 OH

PGE synthase PGH 2 OH Requires GSH PGE 2 OH

Tissue distribution: NARROW PGES MGST 1 WIDE NARROW MGST 2 MGST 3 5 -Lipoxy.

Tissue distribution: NARROW PGES MGST 1 WIDE NARROW MGST 2 MGST 3 5 -Lipoxy. Leukotriene genase C 4 synthase activating protein GSH-dep. oxidoreductase GST: s Glutathione peroxidases

MGST 1 activation

MGST 1 activation

MGST 1 is activated by sulfhydryl reagents SH SH SH SNEMSNEM N + O

MGST 1 is activated by sulfhydryl reagents SH SH SH 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 k. Da) Activation does occur under toxic and oxidative stress in vivo!

Thiolate anion formation is activated Activation increases the rate of thiolate anion formation (not

Thiolate anion formation is activated Activation increases the rate of thiolate anion formation (not the chemical step)

Activation of MGST 1 by reactive intermediates in vivo (2 -3 fold) Diethylmaleate (direct)

Activation of MGST 1 by reactive intermediates in vivo (2 -3 fold) Diethylmaleate (direct) CCl 4 P 450 • CCl 3 Acetaminophen P 450 Reactive quinoneimine

Activation Mechanisms of MGST 1

Activation Mechanisms of MGST 1

Activation of MGST 1 by S-thiolation In vitro by GSSG/GSH ratio = 50 at

Activation of MGST 1 by S-thiolation In vitro by GSSG/GSH ratio = 50 at half maximal activation In vivo by hydroperoxide Sies et al, ABB 322, 288

Capacity and throughput CAPACITY: 0. 2 m. M Glutathione transferase in liver + 5

Capacity and throughput CAPACITY: 0. 2 m. M Glutathione transferase in liver + 5 m. M 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

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

Examples of drugs that are conjugated to GSH • Paracetamol (analgesic, antipyretic) • Carbamazepine (analgesic) • Indomethacin (anti-inflammary)

Paracetamol

Paracetamol

Indomethacin

Indomethacin

The nucleophile substrate Most Aerobic Organisms contain: • m. M concentrations of the low

The nucleophile substrate Most Aerobic Organisms contain: • m. M concentrations of the low molecular weight nucleophile, Glutathione, g-L-Glu-L-Cys-Gly (GSH)