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GenOMeC – SIENA 29/03/13 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 COOH GGT catalytic site Ser 406 Asp 423 Thr 524 plasma membrane NH 2 γ-Glutamyltransferase, γ -Glutamyl transpeptidase (γ-GT; GGT; EC 2.3.2.2.) GGT effects the recovery of extracellular GSH extracellular GSH acceptor gly-cys-SH γ-glu-acceptor GGT GSH (intracellular) DIPEPTIDASES glutamic acid cell glycine intracellular cysteine GSH resynthesis Glutathione, GSH GGT glutamate glycine cysteine H N O II C C N II H O COO- H H3 N+ COO- HS metabolite: Cys-Gly reduced glutathione GSH : SEVEN FACES / FOUR ROLES a Nucleofile a Cofactor of Enzymes an Antioxidant a Metal ion reductant a Prooxidant formation of GS-NO protein S-glutathiolation antioxidant role “prooxidant” role transport of NO ‘protein modulation’ GGT effects the recovery of extracellular GSH extracellular GSH acceptor gly-cys-SH γ-glu-acceptor GGT GSH (intracellular) DIPEPTIDASES glutamic acid cell glycine intracellular cysteine GSH resynthesis CHEMICALLY-INDUCED EXPERIMENTAL LIVER CARCINOGENESIS DEN 2-AAF in diet i.p. 0 1 2 3 4 weeks SACRIFICE partial hepatectomy Cameron R, Kellen J, Kolin A, Malkin A, Farber E - Cancer Res (1978) GGT foci correspond to GSH-dependent lipid peroxidation + exogenous GSH GGT histochemistry LPO histochemistry Pompella et al., Histochem. Cell Biol. (1996) !? GGT / GSH - dependent lipid peroxidation ? Thiols, metal cation reduction and redox cycling Steven D. Aust Utah State University GHS metabolites can play as metal cation reductants gly-cys-SH Fe 3+ REDOX CYCLING . Fe 2+ gly-cys-S H+ 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 ROS Active site of GGT faces outwards U937 cells – Fluorescent detection of 5-nitrosalicylaldehyde precipitation Vis microscopy Fluorescence microscopy Paolicchi et al. (unublished results) A “pro-oxidant” function for GGT γγ-glu-gly γ−glu-gly 22 2 -glu-gly cell cell cell GSH GSH GSH [pKa (SH) = 8.56] [pKa(SH)=8.56] [pKa (SH) = 8.56] . gly-cys-S gly-cys-S. . gly-cys-S GGT GGT GGT thiyl radical thiylradical radical thiyl 2+ Fe Fe Fe2+2+ gly gly222 gly . O O. _ _ 2 2 gly-cys-S gly-cys-S-gly-cys-S gly-cys-SH gly-cys-SH gly-cys-SH HH22O O22 H++ H [pKa (SH) [pKa(SH)=6.4] [pKa (SH)==6.4] 6.4] H ++ H 3+ 3+ Fe Fe Fe 3+ O O22 Pompella et al., 1997–2002 GGT-dependent production of ROS Human hepatoblastoma HepG2 cells HepG2 cells + GSH HepG2 cells + GSH + gly-gly NBT reduction + GGT inhibitor Paolicchi et al. (1998) R-SH . R-S H2O2 . R-S H2O2 . O2 − R-SH . R-S O2 Fe(III) S . SUPEROXIDE Fe(II) thiyl radicals GSH SH H2O2 SH γ SS SS SH γ SS OUTSIDE SH S-S S-S GGT INSIDE SH γ GSH γ-glutamyl cycle INTRACELLULAR EFFECTS Pompella et al. 2002 GGT activity basally down-regulates membrane protein thiols U937 hystiocytoma / MPB-EaFITC reaction / CLS fluorescence microscopy Control GGT inhibition Dominici et al., Free Rad. Biol. Med. (1999) O2 PROTEIN S-THIOLATION Fe(III) S . SUPEROXIDE Fe(II) thiyl radicals GSH SH H2O2 SH γ SS SS SH γ SS OUTSIDE SH S-S S-S GGT INSIDE SH γ GSH γ-glutamyl cycle INTRACELLULAR EFFECTS Pompella et al. 2002 S-Thiolation >>> Modulation of protein function Oxidation of TNFR-1 increases 125I-TNF-α binding affinity Scatchard analysis Dominici et al. (2004) Bound / free 125I-TNFα ratio 0.12 Control GGT stimulation = GGT inhibition increased affinity GGT stimulation 0.10 0.08 0.05 0.03 GGT inhibition = decreased affinity 0.00 0.0020 0.0045 Bound 0.0070 125 0.0095 I-TNFα (nm) 0.0120 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 GGT-dependent redox effects have been documented and published BIOMARKERS GGT protein complexes® Pompella et al., 1997–2011 PAT. PCT/IB2008/052499 - WO2009/001290-A3 GSAO GSNO ACCUMULATION IN ATHEROSCLEROTIC PLAQUES GSH shedding GGT Cis Glu + Cis = ϒ-Glu-Cis GCAO NO release Cys-Gly Cys DIPEPTIDASE METAL REDUCTION REDOX CYCLING −SH OXIDATION TNFR1 ROS Oxidation of AA to DHA increased binding affinity Vitamin C resupply ASK-1 / p38 phosphorylation ADDUCTS with CISPLATIN Gly-Cys-CDDP-Cys-Gly NF-kB H2O2 GSH RESYNTHESIS ANTIOXIDANT ROLE translocation & transactivation induction of CATALASE IL-8 expression SOD CAT DFO BHT Trolox ABBA GENOMIC INSTABILITY CANCER PROGRESSION GGT – bioactivity & pathophysiology • effects in drug resistance: Cisplatin (CDDP) • 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 • MORE… Plant GGT GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT Cisplatin reacts with gly-cys much faster than with GSH cell cell GSH + Cisplatin TOXICITY PERSISTS GGT gly-cys/cisplatin complex H+ gly-cys-S– gly-cys-SH + Cisplatin HN gly-cys-S - S NH Pt gly-cys-S - GSH/cisplatin complex S EXTRACELLULAR DETOXICATION Franzini et al., Eur. J. Cancer (2006) GGT expression confers cisplatin resistance Cell growth Me665/2/60 melanoma cells + Cisplatin 2.5 µM + ABBA Pompella et al. (unpublished results) DRUG ACTIVATION: 4-(N-(S-glutathionyl-acetyl)amino) phenylarsinous acid GSH AO GSAO a promising anti-angiogenic γ-glutamyltransferase cleaved and activated by γ-glutamyltransferase GCAO GGT-expressing cells: more cisplatin-resistant, more GSAO-sensitive high GGT cells high GGT cells effects of GSAO effects of cisplatin Dilda et al. (unpublished results) GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT S-Nitrosoglutathione (GSNO) is the storage and transport form of NO GSNO is a substrate for GGT .NO GGT GGT – bioactivity & pathophysiology • effects in drug resistance: Cisplatin (CDDP) • 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 • MORE… Plant GGT Tissue areas rich in GGT can retrieve .NO from GSNO GSNO .NO .NO GGT . .NO GSNO GSNO GSNO .NO .NO GGT . .NO .NO .NO GGT .NO . NO GSNO NO NO Endothelial GGT mediates the vasorelaxant effect of GSNO in isolated rat aortic rings – Dahboul et al., PLoS One 2011 AORTIC WALL GSNO released NO protein nitrosylation GGT activity GSNO metabolism GSNO alone GSNO + GGT substrates GSNO + GGT inhibition Endothelial GGT mediates the vasorelaxant effect of GSNO in isolated rat aortic rings – Dahboul et al., PLoS One 2011 GGT stimulation % o f relaxation 100 50 GGT inhibition 0 -10 -9 -8 -7 -6 log [GSNO], M -5 -4 Incidence (%) 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) Serum GGT is a factor in utilization of GSNO Bramanti et al., Arch. Biochem. Biophys. (2009) GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT GGT involved in pathogenesis of atherosclerosis? Circulation 112: 2078 -80, 2005 2078-80, Serum and tissue GGT activity occur in the form of four (macro)-molecular complexes 3 4 1 2 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 post-column fluorescent reaction® Franzini et al., Anal. Bioch. 2008 Franzini et al., Clin. Chem. Lab. Med. 2010 Patent pend. PCT2007/IB2008/05499 Sorta srl. a University of Pisa Spin-off www.sorta-biomedical.com GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT GGT is contained in PMN granules FIXED / γ-Glutamyl-4-methoxy-2-naphthylamide + Fast Garnet GBC Corti et al., PLoS One 2013 Activated PMNs release GGT UNFIXED: Control f-MLP activated Corti et al., PLoS One 2013 Released GGT promotes NF-kB activation and IL-8 expression G1 IL-8 expression NF-kB / DNA binding Corti et al., PLoS One 2013 + +G gly2 GT +G SH G1 : +GSH +GGT +G SH C : Control / C1 : +GSH +g l ro l y2 (% of control) Co nt BEAS-2b human bronchial epithelial cells GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT GGT in aortic valve disease: calcification and neoangiogenesis CD68 GGT CD68 GGT Cappelli et al., Atherosclerosis (2010) GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT 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 H2O2 IL-8 secretion DNA damage GGT – bioactivity & pathophysiology • 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 • MORE… Plant GGT GGT in barley root cell wall – Salvage of GSH from the root apoplast Ferretti et al., New Phytologist 181: 115-126, 2008 GGT: THIOL METABOLISM GSH – S-thiolations – S-nitrosoglutathione PATHOGENESIS Drug resistance – Inflammation – CHD – Calcifications – H. pylori BIOMARKER GGT serum isoforms® TARGET Dip. di Ricerca Traslazionale, Università di Pisa A. PAOLICCHI S. DOMINICI A. CORTI M. FRANZINI Dip. di Med. Molecolare, Dip. di Biotecnologie Mediche, Università di Siena E. MAELLARO P. TANGANELLI S. CAPPELLI Istituto Nazionale Tumori, Milano C. GIOMMARELLI F. ZUNINO Lowy Cancer Research Center, UNSW Sidney, Australia P. DILDA P.J. HOGG Facultés de Pharmacie et de Science, Université de Lorraine, Nancy (F) P. LEROY I. LARTAUD A. VISVIKIS Ist. di Chimica dei Composti Organo-Metallici – CNR, Pisa E. BRAMANTI Sorta srl. – a University of Pisa Spin-off With the support of Sorta Srl. www.sorta-biomedical.com How is free iron available for these reactions ? Metal cations are physiologically sequestered in specific transport & storage proteins • PATHOLOGY ! • PHARMACOLOGY ! Complexed iron can be released • Breakdown of hemoglobin by H2O2 liberates iron • Gutteridge JMC, FEBS Lett. 1986 • Puppo et al., Biochem. J. 1988 • Puppo et al., Free Rad. Res. Commun. 1988 • Superoxide induces the release of catalytic iron from ferritin • Bolann et al., Eur. J. Biochem. 1990 • Superoxide induces iron release from Fe-S proteins • Liochev SI, Free Rad. Res. 1996 • Imlay et al., Annu. Rev. Biochem. 2008 • GGT itself reductively releases iron from Ferritin & Transferrin • Paolicchi et al., J. Invest. Med. 1999 • Dominici et al., Cancer Cell. Int. 2003 • Corti et al., Free Rad. Biol. Med. 2005
