Gamma-glutamyltransferase activity: a biochemical factor in cell regulation,

pathogenesis and clinical diagnosis

Alfonso Pompella, MD PhDDipartim. di Ricerca Traslazionale NTMC

Università di Pisa

GenOMeC – SIENA 29/03/13

COOH

NH 2

Ser 406� Asp 423�Thr 524

catalytic site

plasma membrane

γ-Glutamyltransferase, γ -Glutamyl transpeptidase(γ-GT; GGT; EC 2.3.2.2.)

GGT

GGT effects the recovery of extracellular GSH

extracellular GSH

acceptor

gly-cys-SH

γ-glu-acceptor

glycinecysteine

glutamic acid

intracellular GSH resynthesis

GSH(intracellular)

GGT

DIPEPTIDASES

cell

Glutathione, GSH

C

N

H3N+

H

HSCOO-

COO-

O

O

HC

NHII

II

glutamate cysteine glycine

reduced glutathione

GGTGGT

metabolite: Cys-Gly

GSH :GSH : SEVEN FACES / FOUR ROLESSEVEN FACES / FOUR ROLES

antioxidant role� a Nucleofile� a Cofactor of Enzymes� an Antioxidant

transport of NO

‘protein modulation’� protein S-glutathiolation

� formation of GS-NO

“prooxidant” role� a Metal ion reductant

� a Prooxidant

GGT effects the recovery of extracellular GSH

extracellular GSH

acceptor

gly-cys-SH

γ-glu-acceptor

glycinecysteine

glutamic acid

intracellular GSH resynthesis

GSH(intracellular)

GGT

DIPEPTIDASES

cell

0 1 2 3 4 weeks

DENi.p.

2-AAF in diet

partial hepatectomy

SACRIFICE

Cameron R, Kellen J, Kolin A, Malkin A, Farber E - Cancer Res (1978)

CHEMICALLY-INDUCED EXPERIMENTAL LIVER CARCINOGENESIS

GGT foci correspond to GSH-dependent lipid peroxidation

GGT histochemistry

Pompella et al., Histochem. Cell Biol. (1996)

LPO histochemistry

+ exogenous GSH

!?!?GGT / GSH GGT / GSH -- dependent dependent

lipid peroxidation ?lipid peroxidation ?

Thiols, metal cation reduction and redox cycling

Steven D. Aust

Utah State University

gly-cys-SH Fe 3+

gly-cys-S. Fe 2+

H+

GHS metabolites can play as metal cation reductants

A.A. Stark, E. Zeiger, D.A. Pagano

Glutathione metabolism by γ-glutamyl transpeptidase leads to

lipid peroxidation. Carcinogenesis (1993) 14(2): 183-189

Avishay-Abraham Stark

University of Tel Aviv , IL

ROSREDOXCYCLING

Active site of GGT faces outwards

Paolicchi et al. (unublished results)

U937 cells – Fluorescent detection of 5-nitrosalicylaldehyde precipitation

Vis microscopy Fluorescence microscopy

A “pro-oxidant” function for GGT

GGT

cell

gly 2

γγγγ-glu-gly 2 GSH

[pKa (SH) = 8.56]

gly-cys-SH [pKa (SH) = 6.4]

gly-cys-S -

H+

gly-cys-S .thiyl radical

Fe3+

Fe2+

O2

O2

H2O2

H+

_.

GGT

cell

gly 2

γγγγ-glu-gly 2 GSH

[pKa (SH) = 8.56]

gly-cys-SH [pKa (SH) = 6.4]

gly-cys-S -

H+

gly-cys-S .thiyl radical

Fe3+

Fe2+

O2

O2

H2O2

H+

_.

Pompella et al., 1997–2002

cell

GGT

γ−glu-gly2

gly2

gly-cys-SH[pKa(SH)=6.4]

gly-cys-S -

gly-cys-S .thiyl radical

GSH[pKa(SH)=8.56]

Fe3+

Fe2+

GGT-dependent production of ROS

Paolicchi et al. (1998)

NBT reduction

HepG2 cells

+ GSH

HepG2 cells

+ GSH + gly-gly+ GGT inhibitor

Human hepatoblastoma HepG2 cells

RR--SHSH RR--SS..

RR--SS..

HH22OO22

HH22OO22

RR--SHSH

OO22.. −−

RR--SS..

GGT

SH

γγγγ

SH

GSH

γ-glutamyl cycle

SHSS

SS

SS

S.

OUTSIDE

INSIDE

O 2

Fe(III)

Fe(II)

SUPEROXIDE

H2O2

S-S S-S

GSH thiyl radicals

γγγγ

SH

SH

γγγγ

INTRACELLULAR EFFECTS

Pompella et al. 2002

Dominici et al., Free Rad. Biol. Med. (1999)

GGT activity basally down-regulates membrane protein thiols

U937 hystiocytoma / MPB-EaFITC reaction / CLS fluorescence microscopy

Control GGT inhibition

GGT

SH

γγγγ

SH

GSH

γ-glutamyl cycle

SHSS

SS

SS

S.

OUTSIDE

INSIDE

O 2

Fe(III)

Fe(II)

SUPEROXIDE

H2O2

S-S S-S

GSH thiyl radicals

γγγγ

SH

SH

γγγγ

INTRACELLULAR EFFECTS

Pompella et al. 2002

PROTEIN S-THIOLATION

S-Thiolation >>> Modulation of protein functionOxidation of TNFR-1 increases 125I-TNF-α binding affinity

Dominici et al.(2004)

0.00

0.03

0.05

0.08

0.10

0.12

Bou

nd /

free

125 I

-TN

Fα

rat

io

0.0020 0.0045 0.0070 0.0095 0.0120

Bound 125I-TNFα (nm)

GGT stimulation

GGT inhibition

ControlScatchardanalysis

GGT inhibition

=

decreased affinity

GGT stimulation

=

increased affinity

More targets at the cell surface :

GGT prooxidant effects regulate voltage-gated K + channelsZheng MQ et al., Am J Physiol Cell Physiol 297: 253-262, 2009

A number of GGTA number of GGT--dependent redox effects dependent redox effects

have been documented and publishedhave been documented and published

Glu + Cis = ϒϒϒϒ-Glu-Cis

GSH

Cis

CysGSH RESYNTHESIS

ANTIOXIDANT ROLE

GSAO

GCAO

GSNO

NO release

Oxidation of

AA to DHA Vitamin C resupply

GENOMIC INSTABILITY

CANCER

PROGRESSION

GENOMIC INSTABILITY

CANCER

PROGRESSION

ADDUCTS with CISPLATIN

Gly-Cys-CDDP-Cys-Gly

SOD CAT DFO BHT Trolox ABBA

H2O2

ASK-1 / p38phosphorylation

induction of

CATALASE

NF-kBtranslocation

& transactivation

−SH OXIDATIONincreased binding affinity

TNFR1

METAL REDUCTION

REDOX CYCLING

ROS

GGT protein complexes ®

PAT. PCT/IB2008/052499 - WO2009/001290-A3BIOMARKERSBIOMARKERS

shedding

Cys-Gly

GGTGGT

ACCUMULATION IN

ATHEROSCLEROTIC

PLAQUES

IL-8 expression

DIPEPTIDASE

Pompella et al., 1997–2011

•• effects in drug resistance: Cisplatin (CDDP)effects in drug resistance: Cisplatin (CDDP)

•• effects in drug activation: GSAOeffects in drug activation: GSAO

•• GGT catalyzes NO release from GSNOGGT catalyzes NO release from GSNO

•• relationships with vascular tone and hypertensionrelationships with vascular tone and hypertension

•• relationships with atherosclerosis and CHDrelationships with atherosclerosis and CHD

•• relationships with neutrophils and inflammationrelationships with neutrophils and inflammation

•• GGT protein and activation of osteoclastsGGT protein and activation of osteoclasts

•• MOREMORE…… Bacterial GGTBacterial GGT

•• MOREMORE…… Plant GGTPlant GGT

GGT GGT –– bioactivity & pathophysiology bioactivity & pathophysiology

•• effects in drug resistance: Cisplatineffects in drug resistance: Cisplatin

•• effects in drug activation: GSAOeffects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

• MORE… Bacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

cellcellGSH

gly-cys-SH gly-cys-S –H+

GGT

GSH/cisplatin complex

gly-cys/cisplatincomplex

Franzini et al., Eur. J. Cancer (2006)

Cisplatin reacts with gly-cys much faster than with GSH

+ Cisplatin

HN NH

gly-cys-S -

S S

Pt gly-cys-S -

EXTRACELLULAR DETOXICATION

TOXICITY PERSISTS+ Cisplatin

GGT expression confers cisplatin resistance

Pompella et al. (unpublished results)

Me665/2/60 melanoma cells

+ Cisplatin 2.5 µM

+ ABBA

Cel

l gro

wth

DRUG ACTIVATION: 4-(N-(S-glutathionyl-acetyl)amino) phenylarsinous acid

GSAO

a promising anti-angiogenic

cleaved and activatedby γ-glutamyltransferase

GSH AO

GCAO

γ-glutamyltransferase

Dilda et al. (unpublished results)

GGT-expressing cells:

more cisplatin-resistant, more GSAO-sensitive

high GGT cells

high GGT cells

effects of GSAO effects of cisplatin

• effects in drug resistance: Cisplatin

• effects in drug activation: GSAO

•• GGT catalyzes NO release from GSNOGGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

• MORE… Bacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

S-Nitrosoglutathione (GSNO) is the storage and transport form of NO

GSNO is a substrate for GGT

GGTGGT.NO

• effects in drug resistance: Cisplatin (CDDP)

• effects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

•• relationships with vascular tone and hypertensionrelationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

• MORE… Bacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

Tissue areas rich in GGT can retrieve .NO from GSNO

GSNO

GGT

GGT

GGT

GSNO

GSNO

GSNO

.NO

.NO .NO

.NO

.NO

.NO .NO

.NO

.NO

.NO .NO

.NOGSNO

Endothelial GGT mediates the vasorelaxant effect of GSNO in isolated

rat aortic rings – Dahboul et al., PLoS One 2011

GGTactivity

GSNO

metabolism

GSNO

released NO

protein nitrosylation

GSNO

alone

GSNO

+ GGT substratesGSNO

+ GGT inhibition

AORTICWALL

-10 -9 -8 -7 -6 -5 -40

50

100

log [GSNO], M

% o

f re

laxa

tion

GGT

stimulation

GGT

inhibition

Endothelial GGT mediates the vasorelaxant effect of GSNO in isolated

rat aortic rings – Dahboul et al., PLoS One 2011

Serum GGT levels are a favouring factor on the development of

hypertension – DH. Lee et al., J.Hum.Hypertens. (2004)

GGT (U/L)Age(years)

Inci

denc

e (%

)

Serum GGT is a factor in utilization of GSNO

Bramanti et al., Arch. Biochem. Biophys. (2009)

• effects in drug resistance: Cisplatin

• effects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

•• relationships with atherosclerosis and CHDrelationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

• MORE… Bacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

Circulation 112: 2078-80, 2005Circulation Circulation 112: 2078112: 2078--80, 200580, 2005

GGT involved in pathogenesis of atherosclerosis?

Serum and tissue GGT activity occur in the form

of four (macro)-molecular complexes

12

3

4

A HIGH PERFORMANCE GEL FILTRATION CHROMATOGRAPHY METHOD

FOR GAMMA-GT FRACTION ANALYSIS - Franzini et al., Anal. Biochem. 2008

GGT of the future – four macromolecular complexes

Size exclusion chromatography with postSize exclusion chromatography with post--column fluorescent reactioncolumn fluorescent reaction®®

Patent pend. PCT2007/IB2008/05499

Sorta srl.Sorta srl.a University of Pisa Spina University of Pisa Spin--offoff www.sortawww.sorta--biomedical.combiomedical.com

Franzini Franzini et al.,et al., Anal. Bioch. 2008Anal. Bioch. 2008Franzini Franzini et al.,et al., Clin. Chem. Lab. Med. 2010Clin. Chem. Lab. Med. 2010

• effects in drug resistance: Cisplatin

• effects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

•• relationships with neutrophils and inflammationrelationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

• MORE… Bacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

γ-Glutamyl-4-methoxy-2-naphthylamide+ Fast Garnet GBC

FIXED /

GGT is contained in PMN granules

Corti et al., PLoS One 2013

UNFIXED:

Control

f-MLP activated

Activated PMNs release GGT

Corti et al., PLoS One 2013

Released GGT promotes NF-kB activation and IL-8 expression

Contro

l

+GSH

+gly

2+G

SH +

gly2

+GG

T

IL-8 expression(% of control)

C : Control / C1 : +GSHG1 : +GSH +GGT

NFNF--kB / DNAkB / DNAbindingbinding

BEAS-2b

human bronchial

epithelial cells

G1

Corti et al., PLoS One 2013

• effects in drug resistance: Cisplatin

• effects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

•• GGT protein and activation of osteoclastsGGT protein and activation of osteoclasts

• MORE… Bacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

Cappelli et al., Atherosclerosis (2010)

GGT in aortic valve disease: calcification and neoangiogenesis

GGT

GGT

CD68

CD68

• effects in drug resistance: Cisplatin

• effects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

•• MOREMORE…… Bacterial GGTBacterial GGT

• MORE… Plant GGT

GGT – bioactivity & pathophysiology

H2O2

H. pylori GGT-dependent H2O2 generation, NF-kB activation, IL-8 secretion

and DNA damage in AGS gastric cells - Gong et al., Gastroenterology 2010; 139: 564

DNA damageIL-8 secretion

• effects in drug resistance: Cisplatin

• effects in drug activation: GSAO

• GGT catalyzes NO release from GSNO

• relationships with vascular tone and hypertension

• relationships with atherosclerosis and CHD

• relationships with neutrophils and inflammation

• GGT protein and activation of osteoclasts

• MORE… Bacterial GGT

•• MOREMORE…… Plant GGTPlant GGT

GGT – bioactivity & pathophysiology

GGT in barley root cell wall – Salvage of GSH from the root apoplast Ferretti et al., New Phytologist 181: 115-126, 2008

GGT:

THIOL METABOLISMGSH – S-thiolations – S-nitrosoglutathione

PATHOGENESISDrug resistance – Inflammation – CHD – Calcifications – H. pylori

BIOMARKERGGT serum isoforms ®

TARGETTARGET

Dip. di Ricerca Traslazionale ,Università di Pisa

A. PAOLICCHIS. DOMINICI

A. CORTIM. FRANZINI

Dip. di Med. Molecolare,Dip. di Biotecnologie Mediche, Università di Siena

E. MAELLAROP. TANGANELLIS. CAPPELLI

Ist. di Chimica dei Composti Organo-Metallici – CNR, Pisa

E. BRAMANTI

Istituto Nazionale Tumori, Milano

C. GIOMMARELLIF. ZUNINO

Lowy Cancer Research Center, UNSW Sidney, Australia

P. DILDAP.J. HOGG

Facultés de Pharmacie et de Science, Université de Lorraine, Nancy (F)

P. LEROYI. LARTAUDA. VISVIKIS

With the support of Sorta Srl.Sorta srl. – a University of Pisa Spin-off

www.sorta-biomedical.com

Sorta srl. – a University of Pisa Spin-off

www.sorta-biomedical.com

How is free iron available for How is free iron available for

these reactions ?these reactions ?

Metal cations are physiologically sequestered Metal cations are physiologically sequestered

in specific transport & storage proteins in specific transport & storage proteins

•• PATHOLOGY !PATHOLOGY !

•• PHARMACOLOGY !PHARMACOLOGY !

Complexed iron can be released

• GGT itself reductively releases iron from Ferritin & Tran sferrin

• Paolicchi et al., J. Invest. Med. 1999• Dominici et al., Cancer Cell. Int. 2003• Corti et al., Free Rad. Biol. Med. 2005

• Superoxide induces the release of catalytic iron from ferritin

• Bolann et al., Eur. J. Biochem. 1990

• Breakdown of hemoglobin by H 2O2 liberates iron

• Gutteridge JMC, FEBS Lett. 1986 • Puppo et al., Biochem. J. 1988• Puppo et al., Free Rad. Res. Commun. 1988

• Superoxide induces iron release from Fe-S proteins

• Liochev SI, Free Rad. Res. 1996• Imlay et al., Annu. Rev. Biochem. 2008