Báo cáo khoa học: H1-Protein Crosslinking by Transglutaminases docx

H1-Protein Crosslinking by Transglutaminases H1–001 Introduction to the chemistry and biology of transglutaminases (TGs) L. Lorand Cell and Molecular Biology, Northwestern University Feinberg Medical School, Chicago, IL USA. E-mail: l-lorand@northwestern.edu Eukaryotic TGs evolved from the papains, but utilizing a Trp/ Cys/His/Asp catalytic tetrad instead of Gln/Cys/His/Asp for acy- lation-deacylation. Because saturable binding of specific amine substrates, aminolysis is preferred over hydrolysis in deacylation. Transamidation leads to protein remodelling of the gamma amides of select Gln residues by crosslinking to epsilon amines of select Lys’s or by incorporation of small primary amines. Such post-translational modifications have important biological conse- quences. Thrombin/Ca2 + -activated plasma Factor XIII, a mem- ber of the TG family, is essential for stabilizing blood clots; it can also promote angiogenesis. Another form of the factor acti- vated by Ca2 + alone, crosslinks the angiotensin receptor and causes adhesion of monocytes, thereby initiating atherosclerosis in hypertension. In platelets, it catalyzes protein crosslinking and incorporation of serotonin into G proteins to promote release of procoagulants. Other TGs are essential for forming the cornified envelope of skin; stabilize dermo-epidermal junctions; mediate cell-matrix interactions and aid mineralization; bind and hydro- lyze GTP. TGs are involved in autoimmune, inflammatory and degenerative conditions, and apoptosis, while diverse hereditary diseases are caused by the deficiency of a TG (Nature Rev Mol Cell Biol 2003; 4: 140–156). TGs are important catalysts for the synthesis of novel biocompatible polymers, also for incorporation of a variety of bioactive substances, including growth factors. H1–002 Allosteric regulation of the transamidase activity of transglutaminase 2 by guanine nucleotide G. E. Begg 1 , S. E. Iismaa 1 , L. Carrington 2 , S. Holman 1 , A. Husain 1,3 and R. M. Graham 1,4 1 Victor Chang Cardiac Research Institute, Sydney, NSW Australia, 2 School of Mol. and Microbial Sci., University of Queensland, Brisbane, QLD Australia, 3 Physiology Department, University of Alabama, Birmingham, AL, USA, 4 University of NSW, Sydney, NSW Australia. E-mail: b.graham@victorchang.unsw.edu.au Transglutaminase 2 (TG2) is a multifunctional protein, with actions including receptor signalling, extracellular matrix assem- bly and apoptosis. It has two enzyme activities: receptor-stimula- ted signalling that requires GTP binding, and calcium-activated transamidation (TGase activity) that is inhibited by GTP. These activities are associated with distinct conformations induced by the allosteric regulators, calcium and GTP. The recently reported crystal structure of human TG2 bound to GDP identified a novel guanine nucleotide-binding site comprised of residues in the first beta barrel and core domains [Liu, S., Cerione R.A., & Clardy J. Proc Natl Acad Soc USA 2002; 99, 2743–2747]. Here we have investigated the allosteric mechanism by which GTP inhibits TG2 TGase activity. We have shown that of the proposed GTP binding-site residues, S171, F174 and R579 are indeed critical for GTP binding, while R476 and R478 are individually less import- ant. We demonstrate using sedimentation velocity analysis that in the absence of GTP, TG2 undergoes dynamic sampling of expan- ded and compact conformations, and that GTP-binding shifts the equilibrium to a compact conformational substate. This allows a hydrogen bond to form between the active site cysteine in the core domain, and a tyrosine residue in the first beta-barrel domain. We show that this Cys/Tyr bond not only stabilizes the compact conformation, but is critical for inhibition of TGase activity by GTP. Furthermore, we show that R579 is a conform- ational switch residue that, in the absence of GTP, destabilizes the compact conformation. Our findings support a unique model for allosteric regulation of TG2, in which GTP not only masks the destabilizing R579 residue, but also links the core and first beta-barrel domains, thereby stabilizing the compact conforma- tion, facilitating C277/Y516 H-bond formation, and inhibiting substrate access to the active site. H1–003 Chemistry and Biology of Human Transglutaminase 2: its role in celiac sprue and other diseases C. Khosla Departments of Chemistry, Chemical Engineering, and Biochemistry, Stanford University, Stanford, CA, USA. E-mail: khosla@stanford.edu Tissue transglutaminase (a.k.a. transglutaminase 2; TG2) cata- lyzes cross-linking between selected glutamine c-carboxamide groups and lysine e-amino groups in proteins. Although the chemistry of this enzyme is well understood, its multi-faceted role in mammalian biology is only just beginning to be appreciated. TG2 also has considerable medicinal significance, as it is believed to play a crucial role in the pathogenesis of diverse human disor- ders, including celiac sprue, neurological disorders and certain Abstracts 408 types of cancers. We have taken a chemical approach toward understanding the biology and pharmacology of human TG2. Our studies focus on the design, evaluation and application of new substrates and inhibitors of this remarkable enzyme. Of fore- most interest is the role of this enzyme in celiac sprue pathogene- sis. Our recent findings on this subject will be presented. H1–004 Transglutaminase-mediated immobilization of growth factors within fibrin for tissue healing J. A. Hubbell Integrative Biosciences Institute, Ecole Polytechnique Fe ´ de ´ rale de Lausanne, Lausanne, Switzerland. E-mail: jeffrey.hubbell@epfl.ch Growth factor-based therapies have held much promise in tissue repair, but the clinical results of such therapies have not met with initial expectations. One limitation may be in the mode of presen- tation of the factors: in nature, most growth factors are presented bound to extracellular matrix components, and moreover signal- ling involves ligation of both the growth factor receptors and also the extracellular matrix adhesion protein receptors. In contrast to this, most growth factors have been explored in therapy as soluble proteins, substantially unrelated to their natural mode and contact of presentation in development and repair. To begin to address the disconnect between the natural function and therapeutic pres- entation of growth factors, our group has explored the design and production of growth factors, as well as related adhesion mole- cules, capable of binding clinically useful cell invasion matrices. We have focused on fibrin as such as matrix material. Growth fac- tor variants have been designed as fusion proteins of a fibrin-bind- ing immobilization site, a proteolytic cleavage site, and the growth factor of interest. As a fibrin-binding domain, we have selected a factor XIIIa substrate from the N-terminus of alpha 2 plasmin inhibitor, and we have demonstrated that fusion proteins contain- ing this domain are effectively and nearly quantitatively coupled into fibrin during coagulation. Chronic dermal wound may related to an angiogenic deficit in the skin, and for this reason we have addressed the function of several candidate angiogenic growth fac- tors. Vascular endothelial growth factor A 121 (VEGF-A121) was expressed as such a fusion protein, with the transglutaminase sub- strate (TG) at the N-terminus, an adjacent plasmin-sensitive clea- vage site (pl), and a C-terminal growth factor domain. In vivo, this growth factor bound within fibrin was demonstrated to induce angiogenesis that was more localized, more intense, and most importantly more morphologically normal than the free growth factor. For example, in the chicken chorioallantoic membrane, blood vessels were well formed, with a clearly demarcated basal lamella and associated pericytes, whereas free VEGF-A121 induced angiogenesis without these morphological features. Sim- ilar work platelet-derived growth factor variants is under way. H1–005 Transglutaminase 2 in the balance of cell survival and death L. Fe ´ su ¨ s and Z. Szondy Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary. E-mail: fesus@indi.dote.hu Transglutaminase 2 (TG2), a multifunctional enzyme with Ca2 + - dependent protein crosslinking activity and GTP-dependent G protein functions, is often upregulated in cells undergoing apop- tosis. Studies from several laboratories have revealed that in cul- tured cells TG2 may exert both pro- and anti-apoptotic effects depending upon the type of cell, the kind of death stimuli, the intracellular localization of the enzyme and which of its activities is switched on. The majority of data support the notion that transamidation by TG2 can both facilitate and inhibit apoptosis, while the GTP-bound form of the enzyme generally protects cells against death. Recently described biochemical properties of TG2, including its protein disulphide isomerase and protein kinase activities, also may contribute to either survival or death of cells. In vivo studies using TG2 knock out mice confirm the Janus face action of TG2 in the molecular pathways of the apoptotic pro- gram. They have revealed an additional role of TG2: the preven- tion of inflammation, tissue injury and autoimmunity once the apoptosis has already been initiated. This function of TG2 is par- tially achieved by being expressed also in macrophages engulfing and digesting apoptotic cells. TG2 mediated processes participate in the communication between the phagocytic and the apoptotic cells facilitating both death and clearance of dying cells. H1–006 Characterization of transglutaminase and its role in pancreatic b-cell function and survival S. Dookie 1 , N. G. Morgan 2 , M. Griffin 3 and E. A. M. Verderio 1 1 School of Biomedical and Natural Sciences, The Nottingham Trent University, Nottingham, UK, 2 Peninsula Medical School, Plymouth, UK, 3 Aston University, Birmingham, UK. E-mail: shakthi.dookie@ntu.ac.uk Transglutaminases (TGs) are Ca 2+ -dependent enzymes that cata- lyze the formation of protein crosslinks via intermolecular iso- peptide bonds. Previous work has shown that pancreatic islets express functional tissue-type TG (tTG) and recent work under- taken in tTG-deficient mice points to a possible role for tTG in the development of type-II diabetes. Although these data imply that TGs are required for the maintenance of adequate pancre- atic b-cell function, this has never been characterized at the molecular level. Moreover, the recently described survival role for matrix-associated tTG (Verderio et al, JBC, 2003) has not been investigated in islets. Here we show, for the first time, that the predominant form of TG expressed in both a clonal rat insu- linoma b-cell line (BRIN-BD11) and pancreatic islets from differ- ent species is a novel 55 kDa TG protein which may be involved in stimulus-secretion coupling due to increased enzyme activity upon glucose stimulation. Furthermore, this 55 kDa TG protein was not a proteolytic fragment of tTG since its expression was unchanged following inhibition of matrix-associated proteases. A possible protective role for TGs in conditions resembling the dia- betic milieu has also been investigated in vitro, by inducing conditions of glucotoxicity, oxidative stress and lipotoxicity. A pre-conditioned matrix secreted by urinary bladder carcinoma cells (5637), known to support pancreatic b-cell survival, appeared to improve cell spreading and inhibit caspase-3 activity of clonal b-cells when supplemented with exogenous purified tTG. In con- clusion, these findings suggest the predominant expression and function of a novel TG in pancreatic islets but also provides evi- dence for a survival role of matrix tTG, which may be deposited by different cell types surrounding b-cells in vivo. H1–007P Tissue transglutaminase promotes survival of differentiated SH-SY5Y human neuroblastoma cells following exposure to MPP + K. Beck 1 , L. A. De Girolamo 1 , M. Griffin 2 , A. Hargreaves 1 and E. E. Billett 1 1 Interdisciplinary Biomedical Research Centre, School of Biomedi- cal and Natural Sciences, Nottingham Trent University, Notting- ham, UK, 2 School of Life and Health Sciences, Aston University, Birmingham, UK. E-mail: ellen.billett@ntu.ac.uk Tissue transglutaminase (tTG) is a multi-functional, Ca 2+ dependent enzyme, which modifies proteins by transamidation of Abstracts 409 specific polypeptide bound glutamate residues, resulting in pro- tein crosslinking or incorporation of polyamines into substrates. The role of tTG in cell survival and death pathways remains elu- sive, but it has been implicated in the pathology of several neuro- degenerative diseases. MPTP via its active metabolite MPP + induces symptomatic, biochemical and pathological features of Parkinson’s disease in primates, including man. The role of tTG was investigated in pre-differentiated SH-SY5Y cells exposed to cytotoxic concentrations of MPP + (1 and 5 mm) for 24 h. West- ern blot analysis revealed that MPP + treatment significantly reduced tTG protein levels. On the other hand, MPP + dose dependently increased TG transamidating activity, measured by incorporation of a polyamine substrate (EZ-link-5-[biotinamido] pentylamine) into proteins, detected using a Neutravidin, HRP conjugated secondary antibody and ECL. In the presence of a competitive TG inhibitor (putrescine) and a specific, irreversible inhibitor of internal tTG, (R283), MPP + -mediated TG activity was reduced, suggesting it be, in part mediated by tTG activity. To investigate the role of tTG in MPP + -mediated toxicity, the effects of tTG inhibitors on the viability of cells treated with MPP + were assessed using the MTT reduction assay. Results demonstrated that tTG inhibition exacerbated MPP + toxicity, suggesting that tTG promotes survival in this toxicity paradigm. Given that tTG catalysed crosslinks have been found co-localized with alpha-synuclein in Lewy bodies of Parkinsonian brains, fur- ther research into the role of tTG in MPP + -mediated toxicity is warranted. H1–008P An in silico study of substrate preference for transglutaminase 2 E ´ . Cso ˜ sz 1 , P. Bagossi 1 and L. Fe ´ su ¨ s 1 1 Apoptosis Research Group, Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary, 2 Retroviral Biochem- istry Goup, Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary, 3 Apoptosis Research Group, Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary. E-mail: cseva@indi.biochem.dote.hu Transglutaminases catalyze the Ca 2+ -dependent formation of e-amino-c-glutamil isopeptide bonds between the c-carboxamide groups of glutamine residues and e-amino groups of lysines or primary amines. The protein substrates of transglutaminases are numerous, ranging from proteins of the immune system to pro- teins bearing gene regulatory functions. So far the studies of the primary amino acid sequence for known Gln substrate pro- teins have not revealed a consensus sequence for modification by transglutaminase. We have constructed a database of transgl- utaminase substrate proteins (http://www.biochem.dote.hu/ TRANSDAB) which contains structural information about sub- strates of six different transglutaminase types. Using a computer program, we have analyzed the 3D structure of substrate pro- teins, examining the atoms present in a 1.5 nm radius sphere around the C(delta) of each Gln residue. The first detailed study was carried out for tissue transglutaminase (TG2) using crystal structure data of thirteen substrate proteins where the modified Gln residues are known. We also have calculated the radial distribution function (RDF) for pairs of Gln C(delta) and any other type of atom. The RDF values showed differ- ences for substrate and for non-substrate glutamines. These results indicate that the steric and/or electrostatic environment might be different around the substrate and the non-substrate Gln residues of TG2 determining which Gln residue is recog- nized by the enzyme. H1–009P Human cyclo-statherin: specific cyclization of salivary statherin by transglutaminase as a potential early event involved in the formation of oral protein pellicle T. Cabras 1 , R. Inzitari 2 , C. Fanali 2 , C. Olmi 2 , M. Patamia 3 , M. T. Sanna 1 , E. Pisano 1 , B. Giardina 2,3,4 , M. Castagnola 2,3,4 and I. Messana 1 1 Department of Sciences Applied to Biosystems, Cagliari University, Cagliari, Italy, 2 Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy, 3 Institute for the Chemistry of Molecular Recognition, National Research Council (CNR), Rome, Italy, 4 International Scientific Institute Paolo VI, Catholic University, Rome, Italy. E-mail: tcabras@unica.it Statherin is a phospho-peptide of 43 amino acid residues pecu- liar of human saliva with unusual characteristics, such as high content of Tyr, Pro and Gln and a high degree of structural asymmetry. Recently, a significant low level of statherin in sal- iva of subjects affected by pre-cancerous and cancerous lesions of the oral cavity, but not in those affected by tumours of saliv- ary glands, was demonstrated [1]. The pivotal multifunctional role of statherin in the oral cavity could be connected to its involvement in the formation, through protein crosslinking, of a protein network with proline-rich proteins and histatins. This oral protein pellicle could interact with the oral epithelial-cell plasma membrane, contributing to mucosal epithelial flexibility and turnover [2]. The enzyme responsible for the covalent cros- slink formation is an oral transglutaminase (TGA) highly pre- sent in oral epithelial cells [3]. In the present study the reactivity of statherin in the presence of TGA from human oral epithelial cells was investigated by RP-HPLC ESI-ion trap MS. The mass of the reaction product (5363 vs. 5380 amu) evidenced that the early event of the homotypic TGA reaction is the spe- cific formation of a cyclo-statherin. The RP-HPLC ESI-ion trap MS analysis of the proteolytic digest of cyclo-statherin by proteinase K allowed establishing that the intra-molecular cros- slinking involves Lys-6 and Gln-22. Interestingly, a SIM strat- egy performed on different human salivary samples evidenced in some subjects the presence of cyclo-statherin, even though at a concentration near to the limit of detection of the analytical method (about 0.1 lm/l). References 1. Contucci et al., Oral Diseases 2005; 11: 95–99. 2. Bradway et al., Biochem J 1989; 261: 887–896. 3. Bradway et al., Biochem J 1992; 284: 557–564. H1–010P Neural transglutaminase as a potential target for organophosphate toxicity R. Howden 1 , P. L. R. Bonner 1 , M. Griffin 2 and A. J. Hargreaves 1 1 Interdisciplinary Biomedical Research Centre, School of Biomedi- cal and Natural Sciences, Nottingham Trent University, Notting- ham, Nottinghamshire, UK, 2 School of Life and Health Science, Aston University, Birmingham, West Midlands, UK. E-mail: alan.hargreaves@ntu.ac.uk Transglutaminases (TGases) are a group of calcium-dependent enzymes that are responsible for the posttranslational modifica- tion of proteins by the transamidation of polypeptide bound glutamines. The reaction occurs either through the formation of protein crosslinks or the incorporation of polyamines into sub- strate proteins. Of particular interest is the fact that modified Abstracts 410 TGase activity is known to be involved in several neurodegener- ative conditions in mammals. Since chemically induced neuropa- thies, such as organophosphate toxicity, share a number of common features with these disorders, it was of interest to determine whether TGase might also be affected by exposure to organophosphates. Treatment of differentiating N2a cells with the organophosphate phenylsaligenin phosphate (PSP) is known to inhibit the formation of axon-like processes. TGase assays [1] were carried out on extracts of N2a mouse neuroblastoma cells induced to differentiate for 24 h in the presence of 3 lm PSP, using biotin labelled peptide substrates. Both extracts were shown to prefer substrate peptides enriched in glutamine resi- dues. Extracts from PSP treated cells showed a significant increase in TGase activity compared with non-PSP treated con- trols. A similar result was obtained in porcine brain cytosol extracts treated with 3 lm PSP compared to controls. Our find- ings suggest that PSP exposure is able to induce a significant increase in TGase activity in mouse neuroblastoma cells and mammalian brain extracts, and is consistent with the possibility that modified TGase activity may be a key molecular event fol- lowing exposure to PSP. Reference 1. Trigwell, S.M., Lynch, P.T., Griffin, M., Hargreaves, A. J. and Bonner P.L.R. Analytical Biochemistry 2004; 330: 164–166. H1–011P Extracellular matrix changes induced by transglutaminase 2 block angiogenesis and tumor growth R. A. Jones 1 , P. Kotsakis 1 , T. S. Johnson 2 , D. Chau 1 , E. Verderio 1 , S. Ali 1 and M. Griffin 3 1 School of Biomedical and Natural Sciences, Nottingham Trent University, Nottingham, UK, 2 Sheffield Kidney Institute, Northern General Hospital, Sheffield, UK, 3 School of Life and Health Sciences, Aston University, Birmingham, UK. E-mail: richard.jones2@ntu.ac.uk Destabilization of the extracellular matrix (ECM) is known to be important in both tumor invasion and angiogenesis. Here we describe that by promoting ECM accumulation, the protein crosslinking enzyme transglutaminase 2 (TG2) can serve as a potent inhibitor of angiogenesis in vitro and tumor growth in vivo. Administration of active TG2 to in vitro angiogenesis assays suppressed capillary tube formation without causing cel- lular toxicity, whilst inhibition of endogenous enzyme activity had no effect on angiogenesis. The applied TG2 led to the accumulation of a complex ECM that surrounded capillary tubes, resulting in the blockade of capillary growth and the dis- appearance of endothelial cells from vessels. TG2-induced mat- rix accumulation was accompanied by a decreased rate of ECM turnover and an increased resistance to matrix metalloprotein- ase-1. Addition of TG2 to purified collagen I in vitro led to an accelerated rate of collagen fibril formation, which was depend- ent on its protein crosslinking activity. In vivo, mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, prolonged survival, and in some cases tumor regression following intratumor injection of TG2. Immunohisto- chemical and biochemical analysis of TG2-treated tumors revealed fibrotic-like tissue rich in the applied TG2, collagen, TG2-generated a ˚ (a ˜ -glutamyl)lysine isopeptide crosslink, and a lack of organized vasculature. TG2-mediated modulation of the ECM within a tumor to inhibit angiogenesis and promote tumor fibrosis may provide a new approach to solid tumor therapy. H1–012P Coeliac autoantibodies can enhance transamidating and inhibit GTPase activity of tissue transglutaminase R. Kira ´ ly 1 , Z. Vecsei 1 , T. Deme ´ nyi 1 , I. R. Korponay-Szabo ´ 2 and L. Fe ´ su ¨ s 1 1 Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, 2 Heim Pa ´ l Children’s Hospital, Budapest, Hungary. E-mail: kiralyr@indi.biochem.dote.hu Enzyme functions of tissue transglutaminase (TG2) and their modification by anti-TG2 autoantibodies may play a role in mani- festations of coeliac disease, but the effects observed in previous studies have been controversial. We aimed to evaluate the effect of coeliac autoantibodies on transamidation, deamidation and GTPase reactions catalyzed by TG2 by a systematic biochemical approach and in relation to observed clinical presentation type. Effect of antibodies from coeliac patients representing four groups on the activity of human recombinant TG2 was studied using amine-incorporation into solid and immobilized casein and a kin- etic assay. GTPase activity was determined by charcoal method. Coeliac patient antibodies did not have significant inhibitory effect on transamidation/deamidation activity. In contrast, immuno- globulins from patients with severe malabsorption enhanced the reaction velocity to 105.4–242.2% (mean: 189.5%) compared with control antibodies. This activating effect was dose-dependent, and most pronounced with immobilized glutamine-acceptor substrates, and correlated inversely with the basal specific activity of the enzyme. Immunoglobulins purified from the same patients after a gluten-free diet did not affect transamidating activity significantly. GTPase activity of TG2 decreased to 67.0–73.4% in the presence of antibodies from all coeliac groups. In conclusion, an early and severe malabsorptive clinical manifestation of celiac disease is associated with the presence of autoantibody population that enhances transamidation activity of TG2. H1–013P Calreticulin-bound transglutaminase 2 is poorly accessible to celiac autoantibodies I. Korponay-Szabo ´ 1,2 , T. Halttunen 2 , K. Laurila 2 , I. Dahlbom 3 , J. B. Kova ´ cs 4 and M. Ma ¨ ki 2 1 Dept. of Paediatrics, Univ. of Debrecen, Debrecen, Hungary, 2 Paediatric Research Centre, Univ. of Tampere, Tampere, Finland, 3 Research Unit, Pharmacia Diagnostics, Uppsala, Sweden, 4 Dept. of Gastroenterology-Nephrology, Heim Pa ´ l Children ´ s Hospital, Budapest, Hungary. E-mail: ilma.korponay-szabo@uta.fi Background and aims: Tissue transglutaminase (TG2) is a major autoantigen in celiac disease, an autoimmune disorder induced by the ingestion of gliadin-containing cereal foods. Cal- reticulin (CRT), another putative celiac autoantigen, acts as a regulatory subunit for TG2 during receptor signaling. CRT has some homology with gliadin and belongs to molecular chaper- ones. We investigated whether CRT is able to interact directly with TG2 also in vitro and whether this interaction influences autoantigenic properties of TG2 or of both. Methods: Purified CRT (Sigma) was coated to ELISA plates as a sole antigen or as a capture compound for TG2 (Sigma) to establish a sandwich ELISA with a joint CRT-TG2 antigen. Anti-TG2 monoclonal antibodies (MAb) (n = 4) and serum sam- ples of untreated patients with proven gluten-enteropathy (n = 62) were tested with these antigens and with directly coated TG2 antigen as control. Antibody binding to tissue sections rich in TG2 was also investigated by immunofluorescence. Results: CRT bound TG2 in a dose-dependent manner in the presence of Ca ++ . Both free and CRT-bound TG2 were well Abstracts 411 recognized by the MAb ETG 2742, therefore the reactivities of other antibodies were expressed as percentages (AU) of this stand- ard. The MAb CUB7402 specific for the linear epitope 447–478 of TG2 did not bind to the CRT-bound TG2 (2.9 AU vs. 108.3 AU for the free TG2), the other MAbs gave intermediate results. None of the MAbs while 19 of 62 patient samples reacted with CRT alone, which correlated with the high positivity for gliadin anti- bodies. The mean reactivity of patient samples with the joint CRT-TG2 was 19.0 ± 21.5% of that obtained with the free TG2 antigen after background values with CRT had been subtracted. MAb CUB7402 was not able to bind to intracellular TG2 co- localized with CRT in immunofluorescent studies on tissues, while MAb ETG 2742 showed a partial co-localization. Gliadin inter- fered with the CRT-TG2 complexing in a dose-dependent manner. Conclusions: Binding of CRT to TG2 involves the 447–478 ami- noacids of TG2 and results in the masking of antigenic epitopes relevant for celiac autoantibodies. Improper chaperone function or displacement of CRT by gliadin might contribute to the induc- tion of TG2 autoantibodies in celiac disease. H1–014P Proteolytic processing and secretion of Factor XIII in articular cartilage C. M. Gohr and A. K. Rosenthal Department of Rheumatology, Medical College of Wisconsin, Milwaukee, WI, USA. E-mail: akrose@mcw.edu Transglutaminase enzymes (Tgases) have recently been implicated in the pathologic matrix mineralization that occurs in articular cartilage in advanced osteoarthritis. Surprisingly, plasma transgl- utaminase (factor XIII) is present in articular cartilage in addition to the ubiquitous enzyme, tissue (type II) Tgase. We hypothesize that extracellular factor XIII plays a key role in pathologic matrix calcification in osteoarthritic articular cartilage, yet little is known about mechanisms of activation and secretion of cartilage factor XIII. We sought to examine processes of proteolytic activation and extracellular secretion for cartilage factor XII. Immunohisto- chemistry demonstrated high levels of extracellular factor XIII concentrated in areas of matrix mineralization in human osteoar- thritic cartilage. Factor XIII was present in extracellular matrix in cultured chondrocytes and in association with articular cartilage matrix vesicles. These membrane-bound, chondrocyte-derived ves- icles play a key role in matrix mineralization in cartilage. Like fac- tor XIII, chondrocyte Tgase activity was stimulated by calpain and furin as well as thrombin and trypsin. To determine potential mechanisms of factor XIII secretion, chondrocytes were exposed to brefeldin, an inhibitor of classical protein secretion, or methyl- amine, an inhibitor of non-classical protein secretion. Brefeldin caused no change in extracellular matrix factor XIII levels. In con- trast, methylamine dramatically reduced quantities of factor XIII in chondrocyte matrix. These findings support an important role for factor XIII in promoting matrix mineralization in articular cartilage. Understanding the processing and secretion of factor XIII in articular cartilage may lead to the development of novel therapies for osteoarthritis. H1–015P An attempt to identify the active center for protein disulfide isomerase reaction in tissue- type transglutaminase Y. Michiyama, M. Kikuchi, G. Hasegawa and Y. Saito Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan. E-mail: ysaito@bio.titech.ac.jp We have found that tissue-type transglutaminase (tTG), also called as TGc, TGase2, and Ga h , has the activity of protein disulfide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. It was substantially inhibited by the simul- taneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least two hundred times did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalyzed by domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG com- pletely abolished the transglutaminase activity as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca 2+ . It was not inhibited by nucleotides including GTP at all unlike the other activity of tTG. Our results indicated that a disulfide bond(s) might serve as the active site. We have been able to show the presence of the complex made up with the enzyme and the substrate via mixed-disulfide bond(s). We have started the investigation to elucidate the active site disulfide bond. For this matter we synthesized a novel convenient modification reagent for -SH groups. We have not completed the elucidation of the active site, but yet I believe we are very close to the end. H1–016P Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death Z. Sarang 1 , P. Molna ´ r 2 ,T.Ne ´ meth 2 , S. Gomba 2 , T. Kardon 3 , G. Melino 4 , S. Cotecchia 5 ,L.Fe ´ su ¨ s 1 and Z. Szondy 1 1 Apoptosis Signaling Laboratory, Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary, 2 Department of Pathology, University of Debrecen, Debrecen, Hungary, 3 Department of Medical Chemistry, Semmelweis Univer- sity of Medicine, Budapest, Hungary, 4 IDI-IRCCS, University of Tor Vergata, Rome, Italy, 5 Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland. E-mail: sarang@indi.dote.hu TG2 is a protein cross-linking enzyme known to be expressed by hepatocytes, and to be induced during the in vivo hepatic apopto- sis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver dis- eases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-outs within 20 h. While TG2 -/- thymocytes die in the presence of anti-Fas antibodies at the same rate as wild types, TG2 -/- hepatocytes have increased sensitivity towards anti- Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas or the levels of FLIP L , but a decrease in the Bcl-x L expression, and appearance of apoptotic cells with unusual morphology. The same dose of anti-Fas anti- bodies in wild-type mice induced mostly endothelial cell apopto- sis and consequent necrosis in hepatocytes. Administration of chloroethylclonidine, an AR antagonist, to wild-type mice resul- ted in down-regulation of Bcl-x L in the liver, and sensitized hepatocytes for Fas-mediated apoptosis. Accordingly, mice defici- ent in AR expression were also more sensitive to Fas-induced killing, their liver was more sensitive to Fas-mediated apoptosis, and expressed decreased levels of Bcl-x L . In conclusion, our data Abstracts 412 demonstrate that the loss of TG2 sensitizes hepatocytes for Fas- mediated apoptosis, and this is related to an impaired AR signa- ling that regulates the levels of Bcl-x L . Acknowledgment: This study was supported by OTKA T034191, T034 918, ETT 48/2000, ETT 04/605-00 and QLK3- CT-2002-02017. H2–Oxidation of Proteins H2-001 Oxidation of proteins: an overview A. Benedetti Dipartimento di Fisiopatologia, Medicina Sperimentale e Sanita Pubblica, Universita di Siena, Siena, Italy. E-mail: benedetti@unisi.it Chemically speaking, protein oxidation reactions may involve a variety of reactions including SH oxidation, hydroxylation of cer- tain amino acids, and a number of modifications of proteins, which are directly or indirectly mediated by free radicals. In the endoplasmic reticulum, oxidation of -SH groups is a key event in the folding of newly synthesized peptides. This involves luminal oxidases/isomerases (such as PDI and Ero1) and requires an oxi- dative local environment, which, in turn, is maintained by the interplay between such enzymes and membrane specific transport- ers. Hydroxylation of amino acid residues such as prolyl, lysyl and asparaginyl is a process relevant not only for the post-tran- scriptional modification of collagen and other proteins in the endoplasmic reticulum, but also, in the cytosol, for the regulation of hypoxia-inducible transcription factors, which are main actors in certain pathophysiological cellular responses to oxygen. A num- ber of free radical-mediated alterations of cell proteins, often non- enzymatic, have been described in the last decades. They include not only direct oxidation of amino acid residues and of the pro- tein backbone, but also the indirect damage of proteins by lipid peroxidation products and glycation reactions. A huge number of studies reports on the involvement of free radical-induced protein damage in a variety of biopathological conditions (e.g. ageing, neurodegenerative diseases, atherosclerosis, diabetes), nonetheless, the true role for this event remains to be clarified. H2-002 Redox control in the endoplasmic reticulum. R. Sitia Laboratory of Transport and Secretion of Protein, DiBiT, Universita ` Vita-Salute, Milan, Italy. E-mail: sitia.roberto@hsr.it Many secretory proteins contain disulfide bonds that are essential for folding and function. The process of achieving the correct ter- tiary structure and tying it with disulfide bonds occurs in the ER, under the assistance of specialized protein machineries that con- trol the conformity of the result and finally decide the fate of the newly formed protein (quality control). The main oxidative fold- ing pathway requires the transfer of oxidative equivalents to newly synthesized cargo proteins via protein disulfide isomerase (PDI), which in turn is oxidized by Ero1 proteins (Ero1a and Ero1b in mammals, the latter being induced during ER stress). However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and reduction from termin- ally misfolded proteins prior to their dislocation to the cytosol for proteasomal degradation. How is Ero1 activity regulated? We tackled this question following different lines of investigation. In a search for proteins capable of interacting with Ero1, we identi- fied ERp44, a novel multifunctional ER folding assistant. In par- allel, the characterization of a wide panel of Ero1a mutants revealed interesting features in intra-molecular electron transfer. We also demonstrated that import of cytosolic GSH is necessary to balance the oxidative power of Ero1. Ero1a over-expression increases the GSH content in HeLa cells, implying the existence of inter-compartmental regulatory pathways that control redox signaling and homeostasis. Our results shed some light on the molecular networks involved. H2-003 Endoplasmic reticulum lumen: a Janus-faced compartment G. Ba ´ nhegyi Endoplasmic Reticulum Research Group, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary. E-mail: banhegyi@puskin.sote.hu The oxidizing redox environment in the endoplasmic reticulum (ER) lumen is both a pre-requisite and a consequence of the oxi- dative folding of secretory and membrane proteins. Luminal thi- ols (including protein cysteines and glutathione) are present in more oxidized state than the cytosolic ones. While the ratio between reduced and oxidized glutathione is 100:1 in the cytosol, this value is around 1–2:1 in the ER lumen. The redox potentials calculated from these values are -0.24 and -0.18 V, respectively. Generalizing the observations regarding the redox state of thiols found in the ER of cells engaged in protein secretion, now it is supposed that the redox conditions in the ER lumen are oxid- izing. However, several intraluminal reactions have been des- cribed, which require reducing equivalents. Such reactions involve the isomerization of disulfide bonds, the steps of the vita- min K cycle, the biotransformation of several xenogenous ketones and aldehydes and the local activation of cortisone to cortisol. This circumstance suggests that more redox systems exist in the ER lumen. Since their redox state must be different, it can also be suggested that these systems are functionally uncoupled. While molecular oxygen as the ultimate electron acceptor can be used for the direct generation of oxidizing power in the lumen, reducing equivalents need to be transported from the cytosol. The lecture will focus on ER transporters and intraluminal mech- anisms responsible for the support of luminal reducing reactions. H2-004 Cellular oxygen sensing and the regulation of HIF by protein hydroxylation P. J. Ratcliffe Henry Wellcome Building for Molecular Physiology, Oxford, Oxon United Kingdom. E-mail: pjr@well.ox.ac.uk Hypoxia inducible factor (HIF) is an alpha/beta heterodimeric transcriptional complex that plays a key role in directing cellular responses to hypoxia. Recent studies have defined novel oxygen sensitive signal pathways that regulate the activity of HIF by post-translational hydroxylation at specific residues within the alpha-subunits. HIF prolyl hydroxylation governs proteolytic regulation of HIF, whereas HIF asparaginyl hydroxylation modulates interaction with transcriptional co-activators. These hydroxylations are catalyzed by a set of non-haem Fe(II) 2-oxo- glutarate (2OG) dependent dioxygenases. During catalysis, the Abstracts 413 splitting of molecular oxygen is coupled to the hydroxylation of HIF and the oxidative decarboxylation of 2-oxoglutarate to give succinate and CO2. Hydroxylation at two prolyl residues within the central ‘‘degradation domain’’ of HIF-alpha increases the affinity for the von Hippel-Lindau (pVHL) E3 ligase complex by at least three orders of magnitude, thus directing HIF-alpha polypeptides for proteolytic destruction by the ubiquitin/protea- some pathway. Since the HIF hydroxylases have an absolute requirement for molecular oxygen this process is suppressed in hypoxia allowing the HIF-alpha to escape destruction and acti- vate transcription. Co-substrate and co-factor requirements for Fe (II), ascorbate and the Krebs cycle intermediate (2OG) and inducible changes in the cellular abundance of three closely rela- ted HIF prolyl hydroxylases (PHD1–3) provide additional inter- faces with cellular oxygen status that may be important in regulating the oxygen sensitive signal. H2-005 Prolyl 4-hydroxylases, key enzymes in the synthesis of collagens and the response of cells to hypoxia J. Myllyharju Collagen Research Unit, Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland. E-mail: johanna.myllyharju@oulu.fi Collagen prolyl 4-hydroxylases (C-P4Hs) are essential enzymes in collagen synthesis. Three vertebrate C-P4H isoenzymes are currently known. We have recently produced knock-out mice for C-P4Hs I and II. The C-P4H-I -/- mice died at E10.5, while the C-P4H-II -/- mice have no obvious phenotypic abnormalities. The C-P4H-I +/) ; C-P4H-II -/- double mutants, i.e. mice with no C-P4H-II activity and a decreased C-P4H-I activity, are likewise viable but smaller than the wild-type mice. Detailed structure of C-P4H is currently unknown, but we have determined the domain structure of its catalytic a subunit, identified catalytically critical residues in it and solved the structure of its peptide-substrate-binding domain. It was found to belong to tetratricopeptide repeat domains and possess a groove lined by tyrosines, which were shown to be critical for the binding of collagen-like peptide substrates. The response of cells to hypoxia is chiefly mediated by the hypoxia inducible tran- scription factor HIF that upregulates the expression of (e.g.) ery- thropoietin, VEGF and glycolytic enzymes in hypoxic cells. A novel family of P4Hs and a novel asparaginyl hydroxylase (FIH) have recently been found to play a key role in the adaptation of cells to hypoxia by regulating the stability and activity of HIF. We have expressed the three human HIF-P4Hs and FIH as recombin- ant proteins and characterized their catalytic properties. The K m values of the HIF-P4Hs for O 2 were slightly above the concentra- tion of dissolved O 2 in air, thus indicating that they are very effect- ive oxygen sensors. The K m of FIH was distinctly lower, indicating that a larger decrease in O 2 concentration is needed for a significant decrease in FIH activity. We have also analyzed inhibition proper- ties of the HIF hydroxylases. Furthermore, our studies on the sub- strate specificity of the HIF-P4H isoenzymes and FIH indicate that they hydroxylate the three HIF isoforms with varying efficiencies. H2-006 Detection of protein and lipid oxidation in living cells using fluorescent probes D. Sakharov and K. W. Wirtz Bijvoet Institute, Department of Biochemistry of Lipids, Utrecht University, Utrecht, The Netherlands. E-mail: k.w.a.wirtz@chem.uu.nl A novel strategy will be presented for the visualization of ROS- induced protein oxidation in living cells using acetyl-tyramine fluorescein (acetylTyrFluo). This membrane-permeable probe labels intracellular proteins, which become oxidized at tyrosine residues under conditions of oxidative stress in a reaction similar to o,o-dityrosine formation. The fluorescein-labelled proteins can be visualized after gel electrophoresis and subsequent Western blotting using the anti-fluorescein antibody. Identification of labelled proteins were based on mass spectrometry. Exposure of human fibroblasts to hydrogen peroxide (H 2 O 2 ) resulted in the specific labelling of endoplasmic reticulum resident proteins. Photodynamic treatment (PDT) of these cells, loaded with the photosensitizer Hypocrellin A as to generate singlet oxygen resul- ted in the oxidation of a different set proteins. Under the condi- tions, where the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas hydrogen peroxide treat- ment had no effect on cell survival. This suggests that the oxida- tive stress generated by PDT with Hypocrellin A activates apoptotic pathways, which are insensitive to hydrogen peroxide treatment. By using a ratiometric oxidation-sensitive probe, C11-BODIPY581/591 (C11-BO), which reports on lipid peroxida- tion, oxidative stress was visualized by subjecting cells to PDT loaded with a phthalocyanine photosensitizer Pc4. Interestingly, the photosensitizer showed a broad localization in the cytoplasm, whereas the oxidative stress was mostly limited to vesicular peri- nuclear organelles, most likely lysosomes. As a result of PDT lysosomes disappeared, indicating that lysosomes were damaged. We propose that the massive apoptosis observed is associated with the damage to the lysosomes References 1. Van der Vlies D et al. Biochem J 2002; 366: 825–830. 2. Avram D et al. Proteomics 2004; 4: 2397–2407. 3. Sakharov DV et al. Eur J Biochem 2003; 270: 4859–4865. 4. Sakharov DV et al. FEBS Lett 2005. In press. H2-007P Reactivity and functional significance of cysteine residues of mammalian dipeptidyl peptidases III M. Abramic 1 , U. Imaga 1 , M. Osmak 2 ,L.E ` Iicin-Ain 2 , B. Vukelic 1 , K. Vlahovicec 3 and L. Dolovcak 1,4 1 Laboratory of Cellular Biochemistry, Department of Organic Chemistry and Biochemistry, Rudger Boskovic Institute, Zagreb, CROATIA, 2 Department of Molecular Medicine, Rudger Boskovic Institute, Zagreb, CROATIA, 3 Protein Structure and Bioinformat- ics Group, International Centre for Genetic Engineering and Bio- technology, Trieste, ITALY, 4 Department of Molecular Biology, University of Zagreb, Zagreb, CROATIA. E-mail: abramic@irb.hr Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopepti- dase involved in the intracellular protein catabolism of eukaryo- tes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance. The inactivation kin- etics of human and rat DPP III by sulfhydryl reagent p-hydroxy- mercuribenzoate (pHMB) was monitored by determination of the enzyme’s residual activity with fluorimetric detection. Inactivation of this human enzyme exhibited pseudo-first-order kinetics, sug- gesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3520 M-1min-1. Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540 M-1s-1 for slower react- ing sulfhydryl, and 21,855 M-1s-1, for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order Abstracts 414 constants versus reagent concentration. Peptide substrates protec- ted both mammalian DPPs III from inactivation by pHMB. Phy- siological concentrations of biological thiols and H 2 O 2 inactivated the rat DPP III. Human enzyme was resistant to H 2 O 2 attack and less affected by reduced glutathione than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cyt- osols of highly oxygenated rat tissues. These results provide kin- etic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III. H2-008P Thiol oxidase activity of bovine lens aldose reductase modified by copper ion A. Del Corso, M. Cappiello, G. M. De Donatis, F. Gorini and U. Mura Laboratory of Biochemistry, Department of Physiology and Biochemistry, University of Pisa, Pisa, Italy. E-mail: delcorso@dfb.unipi.it Aldose reductase (ALR2), the first enzyme of the polyol path- way, catalyzes the NADPH-dependent reduction of aldoses and aldehydes. Besides its damaging role during diabetes, linked to sorbitol production, ALR2 appears to be involved in the detoxifi- cation of harmful lipid peroxidation products. Despite being not a metal binding protein, aldose reductase is extremely sensitive to Cu(II). The bovine lens enzyme is readily inactivated by stoichio- metric concentrations of the metal through an oxygen independ- ent modification process. The modified enzyme (Cu-ALR2) retains two copper ions and carries a disulfide bond between Cys298 and Cys303. Both copper ions present on the enzyme molecule retain their ability to catalyzes oxidation of thiol com- pounds. When thiols are incubated in the presence of Cu-ALR2, a loss of reduced thiols is observed in a time dependent manner. The rate of thiol oxidation is proportional to the concentration of Cu-ALR2 and depends on the nature of the thiol used and on pH. Among physiological thiols, while GSH and homocysteine appear to be quite insensitive to oxidation, Cys, cysteamine and Cys-Gly are more prone to oxidation, as it occurs when free cop- per is used as catalyst. The metal ions bound to the enzyme appear as effective as free copper in catalyzing the oxidation of different thiols. The only exception appears to be the case of Cys-Gly, whose susceptibility to oxidation increases, when Cu-ALR2 is used as catalyst instead of free copper. Copper che- lation by ALR2 should represent a potentially damaging event contributing to the oxidative damage induced by the metal. H2-009P A radish peroxidase intrinsically stable towards hydrogen peroxide P. Gil 1 , C. Ferreira-Batista 2 , R. Vazquez-Duhalt 1 and B. Valderrama 1 1 Department of Cellular Engineering and Biocatalysis, Institute of Biotechnology, National University of Mexico, Cuernavaca, More- los Mexico, 2 Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National University of Mexico, Cuerna- vaca, Morelos Mexico. E-mail: gil@ibt.unam.mx Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for mul- tiple applications. However, peroxidases are unstable and are readily inactivated by their substrate, hydrogen peroxide. Here we report the identification, biochemical characterization and clo- ning of the gene encoding a novel hydrogen peroxide-tolerant peroxidase isoenzyme isolated from roots of radish (Raphanus sativus) and named Zo peroxidase, after the greek word meaning permanence. Furthermore, we show that the tolerance of Zo per- oxidase towards hydrogen peroxide is an intrinsic property of the enzyme, not due to the presence of catalase activity. H2-010P Thionylation of PKC by reactive sulfur species: disulfide S-monoxide and disulfide S-dioxide F. L. Huang and K. P. Huang Endocrinology and Reproduction Research Branch, NICHD, NIH, Bethesda, Maryland United States of America. E-mail: huangk@mail.nih.gov Disulfide S-monoxide (DSMO) and -dioxide (DSDO) are power- ful signal molecules during oxidative stress. These disulfide S-oxi- des (DSOs) can be generated by oxidation of thiols (glutathione (GSH), captopril (CPSH)) with H 2 O 2 in the presence of iron or methyltrioxorhenium and the formation of DSOs was more effective with free thiols than with their corresponding disulfides, suggesting that sulfenic and sulfinic acids are the intermediates for the formation of these compounds. Both DSMO and DSDO were purified by HPLC and identified by mass spectrometry and they were highly reactive toward thiol to form mixed disulfide. The reaction rate of DSDO was at least an order of magnitude faster than that of DSMO when tested by reaction with 5-merca- pto-2-nitro benzoate. Both DSOs caused dose-dependent inacti- vation of PKC, whose glutathionylation was detected by Western blot with a specific antibody. The IC50 of GS-DSDO-mediated inactivation of PKC (50 ll) was ~ 10-fold less than that of GS-DSMO and mechanistically, the former mainly caused thionylation and the latter both thionylation and polymerization of PKC. DSOs derived from CPSH, CP-DSMO and CP-DSDO were less potent as PKC inhibitors than their corresponding derivatives from GSH, suggesting that the individual DSOs have their distinct reactivities in spite of possessing similar functional groups. Inactivation of PKC by DSOs could be reversed by DTT at low levels of thionylation but became irreversibly inactivated after extensive thionylation. These results suggest that DSOs derived from a variety of thiols constitute a repertoire of signa- ling molecules with distinctive roles in regulating oxidant-medi- ated cellular responses. H2-011P Changes in activity levels of zinc- and non-zinc enzymes in rat brain stem and salivary glands with zinc meal S. Hayashi 1 , Y. Kojima 2 and S. Fujiwara 1 1 Department of Biochemistry, University of Kyushu Dental College, Kitakyushu, Fukuoka Japan , 2 Department of Pediatric Dentistry, University of Kyushu Dental College, Kitakyushu, Fukuoka Japan, 3 Department of Biochemistry, University of Kyushu Dental College, Kitakyushu, Fukuoka Japan. E-mail: haya-sue@mail.kyu-dent.ac.jp D-aspartate oxidase (D-AspO) is a flavoprotein that catalyzes oxidative deamination of D-aspartate to oxaloacetate, H 2 O 2 and NH 3 . D-AspO has been found in many mammal tissues, showing that D-AspO differs from D-amino acid oxidase (DAO) and is located in the peroxisomes of mammal liver and kidneys. In our previous studies, we used rats to study the effects of low intakes of zinc and calcium on the growth and development in tibia metaphysis. However, we have no report on the enzyme for cofactor zinc in rats salivary glands and brain stem. We aimed to produce and eliminate hydrogen peroxide. Twenty, three week- old male rats were divided into four groups and given a diet con- taining different levels of zinc: normal, deficient, low and high, Abstracts 415 respectively for six weeks. Using the methods of sucrose density gradient centrifugation, we examined the activities of alkaline phosphatase and copper, zinc superoxide dismutase (Cu, Zn-SOD) for cofactor zinc enzyme and manganese superoxide dismutase (Mn-SOD), D-AspO and DAO for non-zinc enzyme to assess zinc nutrition in the brain stem and salivary glands. The results indicated that, the activities of Cu, Zn-SOD in the salivary glands were higher than those other tissues, and the highest in the sublingual gland, and significantly decreased in efficient zinc group. The activity of Cu, Zn-SOD in the brain stem slightly increased in high Zn and Zn deficient diet compared with control diet. These results suggest that the activity of Cu, Zn-SOD is sen- sitive to Zn supplementation and deprivation in the sublingual gland and parotid gland. In the brain stem, the activity of DAO slightly increased in Zn deficient diet and D-AspO increased in high Zn diet. In sublingual glands, activity of DAO decreased in high Zn and low Zn diet. The activity of D-AspO increased in sublingual glands and parotid glands in high Zn diet and decreased in sublingual glands and submandibular glands in Zn deficient diet. Implications of the present studies for hydrogen peroxide metabolism were stated above and compared with Zn metabolism. These studies will be discussed, including new evi- dence that aging or death is going through the process of a loss of the capacity to erase active oxygen from the peroxisomes and that life span extension mechanism is depend on the acquisition of the capacity. H2-012P Effect of sodium hypochlorite on isolated human erythrocyte membranes I. V. Halets and V. M. Mazhul 1 Laboratory of Proteomics, Institute of Biophysics and Cellular Engineering, National Academy of Sciences, Minsk, Belarus. E-mail: inessahalets@mail.ru Hypochlorous acid (HOCl) is a powerful oxidant generated in neutrophils and eosinophils. This oxidative agent plays an important role in membrane components damage under inflam- matory conditions. The aim of our study was to investigate the effect of sodium hypochlorite (NaOCl) on slow (millisecond) internal dynamics (ID) of proteins of erythrocyte membranes and accumulation of lipid peroxidation products in them. Room temperature tryptophan phosphorescence (RTTP) analysis had been used as a monitor of protein ID. The lipid peroxidation was studied by determining the amount of thiobarbituric acid reactive (TBAR) products, mainly MDA. The estimation of total membrane SH-groups was realized using the Ellman rea- gent. It has been found that the dependence of both fast (x1) and long (x2) lifetime components of RTTP of erythrocyte membranes on oxidative reagent concentration in the range 0.25–50 mM is given by the curve with peak at 2.7 mM. The considerable increasing of lifetime components of RTTP at con- centrations below of 2.7 mM oxidative reagent correlates with the reducing of total amount membrane SH-groups. The treat- ment of isolated erythrocyte membranes with higher concentra- tions of NaOCl (4.5–50 mM) results in a reduction of x1 and x2 of RTTP. At that in aforementioned range of NaOCl con- centrations characterized by the increasing of x1 and x2of RTTP the level of lipid peroxidation products remains constant. Thus our studies reveals that NaOCl treatment of erythrocyte membranes results in protein modification related to the oxida- tion of SH-groups, restriction of membrane protein structure ID in concentration range 0.25–2.7 mM of NaOCl and accumu- lation of lipid peroxidation products at more higher concentra- tions (9–50 mM) of this oxidative reagent. H2-013P An in vitro study of effect of copper ion on subspecies of lipoprotein(a) in human serum M. Kadkhodaei-Elyaderani 1 and Z. Faezi Zadeh 2 1 Department of Biochemistry, Jundishapoor University of Medical Sciences, Ahwaz, Iran, 2 Thalassaemia Research Centre, Jundishapoor University of Medical Sciences, Ahwaz, Iran. E-mail: Kadkhodaeim@hotmail.com Lipoprotein(a) is one of the plasma lipoprotein that is composed of an LDL core and glycoprotein apo(a). Lp(a) is present in human plasma in four heterogenous subspecies (Lp(a)Lys-, Lp(a)Lys+1, Lp(a)Lys+2, Lp(a)Lys+3). This subclassification is made according to their ability to bind to lysine sepharose. Patho- genicity of Lp(a) as a risk factor for cardiovascular disease, may depend on its lysine binding site [LBS] activity, which imparts a unique function to Lp(a), including a potential to inhibit fibrinoly- sis. Previous studies have shown that Lp(a)Lys+, Lp(a)Lys+2 and Lp(a)Lys+3 have athero-thrombosis properties. Several evi- dences indicate that serum lipoproteins are sensitive to copper- induced oxidation. In this study the effect of copper ion on lysine binding site activity of Lp(a) was investigated. For this purpose four subspecies of Lp(a) were isolated using affinity chromatogra- phy on lysine sepharose from pooled human serum. Serum lipids were oxidized in the presence of 15, 30, 50, 75 and 100 m of CuCl2. Lipid oxidation was measured as conjugated diene forma- tion determined by spectrophotometric method at 245 nm. Four species of Lp(a) in the oxidized serum was isolated. The results showed a concentration-dependent increase in all the Lp(a)Lys+. These data suggest that copper ion induce a chemical modification to lipoprotein(a) that lead to an increase in LBS activity Lp(a) and thus can promote athero-thrombosis. H2-014P Relationships between ascorbic acid, hydroxyproline and collagen, of wound healing in diabetic rats M. Khaksari Haddad 1 , A. R. Rezaizadeh 2 , M. Mardani 3 , G. R. Asadi Karam 4 and M. Mahmoodi 4 1 Department of Physiology, Kerman Medical University, Kerman, Iran, 2 Department of Anatomy, Rafsanjan University of Medical Sciences, Rafsanjan, Iran, 3 Department of Anatomy, Isfahan Univer- sity of Medical Sciences, Isfahan, Iran, 4 Department of Biochemis- try, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. E-mail: khaksar38@yahoo.co.uk It was reported that increased oxidative stress in diabetes mellitus associated with decrease in collagen synthesis. In this present study, the role of ascorbic acid on hydroxyproline and collagen in diabetic rats is reported. This study was performed on 160 male rats. Diabetes was induced by injection of streptozotocin [STZ, S.C , 55 mg/kg].Animals were divided into four groups, and then rats were remained diabetic for 8 weeks. Control group (I), received the vitamin c deficient diet. Prophylaxia group (II), received 250 mg/lit vitamin c, one month before induction of dia- betes. Treatment group (III), received vitamin c after induction of diabetes. Combination group (IV), received the vitamin c one month before diabetic induction, until wound healing. Full thick- ness incision induced on back skin of rat, 8 weeks after diabetic induction. Content of collagen and biochemical measurement of hydroxyproline at days 1, 3, 7, 11.15, and 20 after operation were measured. The results showed, the mean of collagen content and hydroxyproline in group I was more than all examined groups, at days of 11, 15 and 20, in addition, in group III this indication was less than group IV during all days. This data suggest that, vitamin c supplementation in chronic diabetic rats impaired Abstracts 416 wound healing, and decreased collagen and hydroxyproline con- tent, while used as prophylaxis or combination. H2-015P Oxidative modulation of macromolecular interactions within the cell surface-activated kinin-generating system A. Kozik, M. Rapala-Kozik, U. Blaszczyk and I. Guevara-Lora Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland. E-mail: akozik@mol.uj.edu.pl Bradykinin and related peptides, collectively known as kinins, regulate many physiological processes and are involved in virtu- ally all inflammatory responses. Kinins are excised from precur- sor proteins, kininogens, due to the proteolytic action of kallikreins. The plasma kinin-generating system, called the con- tact system, includes a high molecular mass form of kininogen (HMWK), prekallikrein (PK) and factor XII, and is activated on negatively-charged cell surfaces. In this work, we tested a hypo- thesis that during inflammatory episodes the macromolecular interactions within the contact system may be modified due to the action of reactive oxygen species. Therefore, we oxidized sol- uble components of this system, HMWK, PK and factor XII, and studied their assembly and activation on the surfaces of var- ious cells (neutrophils, macrophages etc.). For protein oxidation, we used the neutrophil myeloperoxidase-hydrogen peroxide- chloride system. The interactions between soluble components of the contact system was studied by high-performance gel filtration, a microplate-binding assay, time-resolved fluorescence polariza- tion measurements and steady-state fluorescence energy transfer measurements. A destructive effect of oxidation on the complex formation was observed for any pair of interacting proteins. Especially, the oxidized HMWK completely lost the ability to bind PK, apparently due to the modification of methionine and tryptophan residues at the PK-binding site. Hence, the oxidized HMWK when bound to the cell surface could not be effectively processed+ after subsequent attachment of PK and factor XII. Hence, the kinin production was quenched. This may happen in real inflammatory foci unless some other proteinases take over the kallikrein role. H2-016P Evaluation of antioxidant activity of tomato treated with an ethylene inhibitor M. A. Murcia, M. Martı ´ nez-Tome ´ and A. M. Jime ´ nez-Monreal Department of Food Science and Technology, Faculty of Veterinary, Universidad de Murcia, Murcia, Spain. E-mail: mamurcia@um.es The majority of antioxidants have a phenolic structure and var- ious phenolic compounds with antioxidative activity and present in the various plant tissues. Tomato contains several antioxidants that could be important in the diet; they are the main foodstuffs contributing to the dietary intake of lycopene. Lycopene is able to act as an antioxidant and scavenger of free radicals, being more effective than carotenes. Prolonging the shelf life of fresh vegetables is a very important concern for both consumers and food packers. Factors affecting the surface colour are related mainly to packaging methods, light exposure, time/temperature regime during storage and the presence of oxidants and antioxi- dants. 1-Methyl cyclopropene is an ethylene inhibitor, which is used as a delayer of senescence of the tomato and we have assayed the antioxidant properties of tomato treated with this ethylene inhibitor, by using the deoxyribose assay. All tomatoes analyzed (treated or not with the ethylene inhibitor) exhibited better antioxidant activities than some common food additives analyzed such as BHA and BHT. Results demonstrated that the treatment of tomatoes with the ethylene inhibitor did not signifi- cantly affect to their antioxidant activity. H2-017P L-Glutamine effect on liver citrulline level J. I. Nikolic 1 , D. T. Sokolovic 1 and B. J. Djindjic 2 1 Department of Biochemistry, University of Nis, Nis, Serbia and Montenegro, 2 Department of Pathological Physiology, University of Nis, Nis, Serbia and Montenegro. E-mail: nikdan@bankerinter.net Trauma, systemic inflammation, sepsis or toxins can modulate tissue functions. Glutamine has been shown to have beneficial effects in sepsis, trauma, infections, chemotherapy etc. The aim of this study was to investigate the possible beneficial effect of L-glutamine on acute hepatotoxicity induced by mercury chloride (HgCl2). To study possible role of nitric oxide in mechanisms toxicity of mercury chloride we have study interaction between L-Glutamine and nitric oxide synthesis in tissue metabolism. Male Spraque-Dawley rats weighing about 200 g. were used in the experiment. Acute toxicity was induced by i.p. administration of mercury chloride in a dose of 3 mg/kg. One group of animals was pre-treated with L-glutamine (200 mg/kg) 1 h before mercury chloride administration. Control group of animals was treated with equal volume of saline. The animals were killed 24 h after HgCl 2 administration. The level of citrulline, by product in nitric oxide synthesis, was measured by diacetylmonoxime reaction. Results of the study show that acute toxicity of mercury chloride leads to elevation of tissue level of citrulline compared to control group (P < 0.01). Administration of glutamine to control rats decreases slowly citrulline level compared to control, while pre- treatment of animals with glutamine before mercury chloride administration leads to the most greater decreases in citrulline level (P < 0.01). Heaving in mind that overproduction of nitric oxide has cytotoxic effects administration of L-glutamine may be beneficial in mercury toxicity through decreasing nitric oxide level. H2-018P Peptides are formed when RNAase is treated with Fenton reagent in the presence of copper (II) V. R. Oso ´ rio e Castro 1 and G. B. Domont 2 1 Laboratory of Biochemistry, Department of Biosciences, Politechnic Institute of Castelo Branco, Castelo Branco, Beira Interior Portugal, 2 Laboratory of Biochemistry, Department of Biochemistry, Federal University of Rio Janeiro, Rio de Janeiro, Rio de Janeiro Brazil. E-mail: valdemar@esa.ipcb.pt The inactivation of RNAase by Fenton reaction, within certain conditions, shows that after 30 min of reaction 22% of the ori- ginal activity is lost. Two active fractions are separated, by filtra- tion of the reaction mixture through Sephadex G-75 columns, suggesting that the smaller active fraction is formed as the result of Fenton reaction. However, the inactivation increases in the presence of copper (II). For example, with 4.4 x 10 –4 Cu ++ it reaches 92% after 1 min and 98% after 30 min of reaction, showing three fractions (F 1 Cu, F 2 Cu, F 3 Cu) by Sephadex G-75 gel filtration. Electrophoretic methods reveal that RNAase, modi- fied by free oxygen radicals, from Fenton reactive, has an elec- trophoretic mobility lower than the control, suggesting a decrease of positive charges on its surface. On the other hand, they also indicate peptides in fractions F 1 Cu and F 2 Cu. Particularly F 2 Cu have, at least, seven peptides, well separated by high voltage Abstracts 417 [...]... by 15% (P < 0.01) and by 30% (P < 0.001) after 2 h and 5 h of forced swimming, respectively TAS was decreased by 20–25% after 2 h or 5 h of exercise AChE was inhibited by 30% (P < 0.001) and 45% (P < 0.001) after 2 h and 5 h of forced swimming, respectively In contrast, (Na+, K+)-ATPase and Mg2+-ATPase were stimulated by 80% (P < 0.001) and 40% (P < 0.001), respectively, after 2 h of swimming and by. .. hepatotoxicity induced by heroin Methods: A dose-increasing method was used to develop the heroin-dependent model in mice The content of malondialdehyde (MDA) was measured by thiobarbituric acid (TBA) method, protein carbonyl by DNPH reagent, reduced glutathione (GSH) by DTNB method and percentage of cell with damaged DNA by comet method, and the activity of alanine aminotransferase (ALT) by commercial kit... transductions by secreted factors from SkMCs infected with bFGF gene, HUVEC was cultured in the Ad/bFGF infected conditioned medium, basal medium containing bFGF or VEGF Raf-1 activation (Tyr340/341 phosphorylation) was induced by conditioned medium or VEGF treatment but not by bFGF Ser338 of Raf-1 was also phosphorylated by conditioned medium or bFGF treatment but not by VEGF These Raf-1 activations by conditioned... cysteine-sulfinic acid form, eventually reversed by ATPdependent reduction by sulfiredoxin Conservation of these mechanisms in higher eukaryotes will be addressed These mechanisms illustrate the built-in high specificity of cysteine-based H2O2 redox signaling and suggest the existence of specific pathways of cysteine oxidation by H2O2 H3-005 Calcium release mediated by redox activation of ryanodine receptors... novel role for protein glycosylation, namely the protection of protein against the attack by radicals generated from a one-electron oxidation of the xenobiotics Acknowledgments: This work was supported by Ministry of Education of Czech Republic (MSM 0021620808), by the Institutional Research Concept (AVOZ5020963), and by the Grant Agency of the Academy of Sciences of Czech Republic (A5020403) References... NB4 cells died by apoptosis and/or necrosis while K562 showed a resistance to apoptosis at all time-periods and DQA concentrations assayed and cell death was always by necrosis This apoptotic resistance may be reversed in the presence of the bcr/abl inhibitor protein STI571 In both cell lines, the cell death seems to be mediated by early alterations of mitochondrial function as evidenced by loss of mitochondrial... protection when present for 23 h prior to 4 h in basal medium, followed by 1 h of H2O2 stress This 6AN-mediated protective effect was associated with a modest increase (by up to 55%) of the cytosolic free Ca2+, and a decrease of total cell GSH down to 56% This protective effect was blocked by inhibition of ryanodine receptors, as well as by inhibition of Ca2+-independent isoforms of PKC The longest of the... evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG EGCG induced activation of p38 mitogenactivated protein kinase (p38... differentiation of HL-60 cells induced by diallyl disulfide (DADS), to explore molecular mechanism in the differentiation of HL-60 cells induced by DADS Methods: Cell differentiation was validated by morphological method and nitroblue tetrazolium (NBT) reduction test Cells were processed for two-dimensional (2-D) electrophoresis Cellular proteins were separated by isoelectric focusing via immobilized... cause the higher RyR2 activity found in these conditions Acknowledgments: This work was supported by Fondecyt grants 1030446, 1030449 and FONDAP 15010006 423 Abstracts H3-015P The activity of 20S proteasome from the yeast S cerevisiae is modulated by redox mechanisms including S-thionylation and reduction by glutaredoxin2 and cytosolic thioredoxins ´ M Demasi1, G M Silva2, F P Cavalher2, J A Barcena3, . H1-Protein Crosslinking by Transglutaminases H1–001 Introduction to the chemistry and biology of transglutaminases (TGs) L remodelling of the gamma amides of select Gln residues by crosslinking to epsilon amines of select Lys’s or by incorporation of small primary amines. Such post-translational