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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