Gamma-glutamyltransferase activity: a biochemical factor ... · PDF file...

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

    pathogenesis and clinical diagnosis

    Alfonso Pompella, MD PhD Dipartim. 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

    glycine cysteine

    glutamic acid

    intracellular GSH resynthesis

    GSH (intracellular)

    GGT

    DIPEPTIDASES

    cell

  • Glutathione, GSH

  • C

    N

    H 3 N+

    H

    HSCOO-

    COO-

    O

    O

    H C

    N HII

    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

    glycine cysteine

    glutamic acid

    intracellular GSH resynthesis

    GSH (intracellular)

    GGT

    DIPEPTIDASES

    cell

  • 0 1 2 3 4 weeks

    DEN i.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

    O 2

    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

    O 2

    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 function Oxidation 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

    B ou

    nd /

    fr ee

    12 5 I

    -T N

    F α

    r at

    io

    0.0020 0.0045 0.0070 0.0095 0.0120

    Bound 125I-TNFα (nm)

    GGT stimulation

    GGT inhibition

    ControlScatchard analysis

    GGT inhibition

    =

    decreased affinity

    GGT stimulation

    =

    increased affinity

  • More targets at the cell surface :

    GGT prooxidant effects regulate voltage-gated K + channels Zheng 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

    Cys GSH 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 / p38 phosphorylation

    induction of

    CATALASE

    NF-kB translocation

    & transactivation

    −SH OXIDATION increased 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

  • cellcell GSH

    gly-cys-SH gly-cys-S – H+

    GGT

    GSH/cisplatin complex

    gly-cys/cisplatin complex

    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

    C el

    l g ro

    w th

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

    GSAO

    a promising anti-angiogenic

    cleaved and activated by γ-glutamyltransferase

    GSH AO

    GCAO

    γ-g