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YSF – Young Scientist Forum

YSF – Young Scientist Forum YSF.1 CV-IIL lectin: another piece of the Chromobacterium violaceum puzzle L. Adamova´1, J. Adam1 and M. Wimmerova´1,2 1 National Centre for Biomolecular Research, Kotla´rˇska´ 2, Brno, Czech Republic, 2Department of Biochemistry, Masaryk University, Kotla´rˇska´ 2, Brno, Czech Republic One ot the most representative (sub)tropical soil microbe, Chromobacterium violaceum, produces many important substances and has great potential for biotechnology and medicine. On the other hand, it can also act as an opportunistic human pathogen. The bacterium produces the lectin CV-IIL, that can help the adhesion bacterium to saccharide moieties at host cells¢ surface. This step is essential for enabling the colonization of host tissue by the bacteria in organism and therefore for starting the infection. Understanding the principle of the interaction can be helpful in designing anti-adhesion therapy. CV-IIL belongs to the PA-IIL lectin family, which includes structurally similar lectins from several other pathogenic bacteria. These lectins have increased affinity for monosaccharides thanks to two calcium ions in the binding site that mediate the sugar-lectin interaction. The CV-IIL lectin has a comparable affinity to fucose and mannose (the other lectins strongly prefer one or the other), and therefore it is ideal for testing of specificity change by mutation. This work is focused on the important amino acid triad 22–23-24, which is suspected to play a decisive role in directing the specificity. Mutants of CV-IIL were prepared by in silico and in vitro directed mutagenesis method, their binding properties were tested by surface plasmon resonance and isothermal titration calorimetry. Complemented with molecular docking, a powerful molecular modeling method, the results of the study offer important additional insight into the structural reasoning behind the sugar preference, as well as provide another part of the wider picture of this important bacteria and its behavior. This work is supported by Ministry of Education (MSM0021622413, LC06030) and Grant Agency of the Czech Republic (GA303/09/1168).

YSF.2 (S14.2.5) Synphilin-1 inhibits alpha-synuclein degradation by the proteasome B. Alvarez-Castelao and J. G. Castanõ Departamento de Bioquı´mica, Instituto de Investigaciones Biome´dicas ‘‘Alberto Sols’’, Universidad Autońoma de Madrid y Consejo Superior de Investigaciones Cientı´ficas (UAM-CSIC), Centro de Investigacioń Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Idipaz, Facultad de Medicina UAM, Madrid, Spain Intracellular deposits of aggregated alpha-synuclein are a hallmark of Parkinson’s disease (PD), the causes involved in the development of these deposits remains unknown, but this fact could be explained by a failure of alpha-synuclein proteostasis. Protein–protein interactions are critical in the regulation of cell proteostasis, a large number of proteins have been described to potentially interact with alpha-synuclein in the cell. Sinphilin-1 interacts both in vitro and in vivo with alpha-synuclein, promoting the aggregation of alpha-synuclein into inclusions containing both proteins. We report here that synphilin-1 specifically inhibits the degradation of alpha-synuclein wild-type and its missense 446

mutants (A30P, E46K and A53T) by the 20S proteasome, due at least in part by the interaction of the ankyrin and coiled-coil domains of synphilin-1 (aminoacids 331–555) with the N-terminal region (amino acids 1–60) of alpha-synuclein. Co-expression of synphilin-1 and alpha-synuclein wild-type in HeLa and N2A cells produces a specific increase in the half-life of alpha-synuclein, as degradation of unstable fluorescent reporters is not affected. This new regulatory mechanism of alpha-synuclein degradation could be important in those tissues that express both proteins, like brain and muscle. Based on these observations one can predict that decreasing synphilin-1 expression levels should relieve its inhibition of alpha-synuclein degradation, Siah-1 is an ubiquitin ligase that targets synphilin-1 to 26S proteasomal degradation, we show that the inhibition of alpha-synuclein degradation caused by synphilin-1 can be relieved by co-expression of Siah-1 in cells. These mechanistic insights into regulation of alpha-synuclein degradation by synphilin-1, that inhibits the proteasomal pathway of degradation of alpha-synuclein and its relieve by Siah-1, may help to understand the pathophysiological changes occurring in PD and other synucleinopathies.

YSF.3 Participation of FABPs in the neurotrophic effect of oleic acid during the postnatal development of the brain A. A. Arroyo-Martin, A. Tabernero and J. M. Medina Department of Biochemistry and Molecular Biology, University of Salamanca Our previous works have shown that oleic acid behaves as a neurotrophic factor for neurons in culture. In fact, oleic acid promotes axonal and dendritic growth and the expression of the axonal growth-associated protein 43 (GAP43) and of the microtubule-associated protein 2 (MAP2). Our last results to date show that albumin promotes the extension of GAP43-positive axons in the postnatal striatum presumably by increasing the amount of oleic acid synthesized by SCD-1 in the periventricular zone and subsequently by carrying the fatty acid to neurons. Oleic acid requires specifically albumin as an extra-cellular carrier to exert its function as a neurotrophic factor because oleic acid alone only slightly up-regulates GAP43 expression and other extracellular fatty acid carrier proteins such as alpha-fetoprotein does not exert any effect. But we have not any information about the intracellular carrier of oleic acid inside the neurons, and this is the aim of this work. This work tries to study the role of a family of intracellular carrier proteins, the fatty acid binding-proteins (FABPs). We propose that these molecules are involved in the axonogenesis promoted by oleic acid. We have analysed by inmunohistochemistry the presence of these proteins in neurons of the striatum and the subventricular zone of newborn rats, in addition we mesured by Western blot the changes of their levels of expression when the oleic acid is present or not. Besides, we have silenced the FABPs isoforms by specific siRNAs in neuronal cultures and in organotypic cultures. Our results show that FABP7 is present at the SVZ and its silencing downregulates the expression of GAP43 and MAP2; FABP5 showed to be essential for the neuronal survival, and FABP3 was localized initially in progenitors of the SVZ and later, in neurons of the striatum when these cells increase their dedritic proyections by oleic acid. In brief, our results indicate that FABPs play a role in the neurotrophic effect of oleic acid.

FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies

YSF – Young Scientist Forum YSF.4 Plasma membrane associated glycohydrolases modulation in Gaucher disease M. Aureli1, N. Loberto1, R. Bassi1, M. Filocamo2, V. Chigorno1, A. Prinetti1 and S. Sonnino1 1 Departement of Medical Chemistry, Biochemistry and Biotecnology for the Medicine, University of Milano, 2S.S.D. Laboratorio Diagnosi Pre-Postnatale Malattie Metaboliche, IRCCS G. Gaslini Glycosphingolipids (GSL) are amphiphilic components of the outer layer of the plasma membranes acting not only a structural role but also as regulators of several proteins associated to the cell plasma membranes (PM) and, together with the PM proteins, they play an active role in the ‘‘cell social life’’. For several years in literature, beside to the complex intracellular sphingolipid metabolism, enzymes able to induce structural changes in the hydrophilic portions of the glycosphingolipid working directly at the cell surface have been described. Our experiments on PM associated glycohydrolases showed a surprisingly high conservation among all the different cell line tested of the activities assayed. In particular, in patient affected by sphingolipidoses, we found for the enzyme involved in the pathology also a reduction of its PM associated activity. However comparing the total and PM activities in normal and pathological cells, we found that the residual activity in the PM was higher than that recovered in the total cells. In particular in fibroblasts deriving from Gaucher disease type 1, 2 and 3 patients, we found that the CBE-sensitive b-glucosidase activity associated to the cell PM resulted down-regulated, whereas GBA2 resulted up-regulated with respect to the normal fibroblast. Moreover other PM associated activities resulted modulated, as b-hexosaminidases, b-galactosidases and arylsulphatases. Among the three sub-types of the Gaucher disease we found a different enzymatic profile of these PM associated activities. Due to the absence of biochemical or biomolecular prognostic assay, we considered this information as the starting point to design new prognostic strategies for the different sub-type of Gaucher disease.

YSF.5 Potential glioma markers Chitinase 3-like 1 and Chitinase 3-like 2 proteins activate MAPK and PI3K/Akt signaling pathways to cause distinct outcomes S. Avdieiev, P. Areshkov and V. Kavsan Institute of Molecular Biology and Genetics, Kiev, Ukraine In an effort to identify genes, which might be used as molecular markers for glial tumors, we compared gene expression in glioblastoma and normal adult human brain. Serial Analysis of Gene Expression found Chitinase 3-like 1 (CHI3L1, HC gp-39, YKL40) and Chitinase 3-like 2 (CHI3L2, YKL-39) genes among the most abundant transcripts in glioblastoma. It was reported that CHI3L1 stimulated DNA synthesis and proliferation by activation of extracellular signal-regulated kinases ERK1/2 (MAPK)and Akt (PI3K)- mediated signaling cascades. Western-blot analysis of 293 cells and glioblastoma derived U373 cells after treatment with CHI3L2 showed increased phosphorylation level of these kinases. Unexpectedly, dose dependent decrease in total DNA content and [3H]thymidine incorporation were observed in 293 cells treated with CHI3L2. Previously was demonstrated that stimulation with EGF gave short activation of ERK1/2 leading to a proliferative signal, whereas treatment with nerve growth

Abstracts factor (NGF) in PC12 cells gave the sustained activation of the pathway that lead to differentiation. CHI3L2 induced sustained phosphorylation of ERK1/2 and Akt in 293 and U373 cells, associated with their nuclear translocation, in a very similar way to that was shown for NGF, while CHI3L1 revealed more transient effect, which was more likely to EGF. These data suggest that CHI3L2 inhibits mitogenesis and may cause differentiation phenotype, but not proliferation. Thus, two novel potential markers of astrocytic gliomas, CHI3L1 and CHI3L2, both trigger ERK1/ 2 and Akt signaling cascades, however, this activation lead to different physiological response of the cell depending on the strength and duration of the signal.

YSF.6 Genistein-induced apoptosis of HL-60 cells via effecting human telomerase reverse transcriptase activity T. Balci, C. B. Avci, S. Yilmaz, Z. O. D. Sigva, M. Yucebas, S. N. Gunduz, C. Kayabasi and C. Gunduz Medical Biology Department, Ege University Medical School, _ Izmir, Turkey hTERT is a susceptibility gene for development of many cancers, including leukemia. Genistein is a strong tyrosine kinase inhibitor, exhibiting estrogen receptor binding activation, DNA topoisomerase II inhibition, and alteration of cell cycle regulatory proteins leading to G2/M arrest; down-regulation of the PI3K/ Akt and NF-kB signalling pathways and activation or inhibition of MAP kinases. Genistein was reported to induce in vitro differentiation in several tumor cell models, including leukemia cells. However, the regulation and signaling of genistein-induced leukemia cell differentiation are poorly known. In this study, the effect of genistein on apoptosis and telomerase activity on HL-60 cells is investigated in time and dose dependent manner. Cytotoxicity of genistein in HL–60 cells was assessed by Trypan Blue Dye and XTT tests at 1–150 lM doses. Annexin V-EGFP method was used to detect apoptosis. The expression analysis of hTERT gene was carried out by using Lightcycler TeloTAGGG hTERT Quantification Kit,. IC50 dose of genistein was found as 50 lM in HL-60 cells. As we assessed the apoptotic effects of genistein in IC50 dose, apoptosis was induced 4.25 times higher, when compared to genistein-free cells, used as the control group. The hTERT activity in genistein-treated HL-60 cells were found to be 5.16, 3.81 and 5.04 times lower in 24, 48 and 72nd hours, when compared to control group. A distinct increase in apoptosis and the reduction of telomerase activity were observed following the treatment of leukemia cells with 50 lM of genistein. Induced apoptosis of HL-60 cells via effecting human telomerase reverse transcriptase activity can be used in the prevention and treatment of leukemia, however further studies are needed to elucidate the molecular mechanism of genistein and telomerase activity.

YSF.7 The inhibition of A328F butyrylcholinesterase mutant by nile blue K. Biberoglu and O¨. Tacal Hacettepe University Mammals contains two main forms of cholinesterases: acetylcholinesterase (AChE, E.C. 3.1.1.7) and butyrylcholinesterase (BChE, E.C. 3.1.1.8). BChE is distinguished from AChE by substrate preferences and differential sensitivity to certain inhibitors It is toxicologically and pharmacologically important in scaveng-


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Abstracts ing and detoxifying cocain, organophosphorus pesticides, carbamate pesticides and chemical warfare agents. In our laboratory, earlier kinetic studies have shown that phenoxazine dyes are highly effective as inhibitors of human and horse BChEs with Ki in the nanomolar -micromolar range. In the present study, aiming to identify the binding sites of the phenoxazine dyes and their functional contacts with the active site, we examined the role of Ala328 in the inhibition of human BChE by nile blue (NB), a phenoxazine dye. The inhibitory effects of NB on recombinant wild type and A328F BChE mutant were studied spectrophotometrically at 25 oC in 50 mM MOPS buffer pH 8, using butyrylthiocholine as substrate. NB caused complex, nonlinear inhibition of wild type BChE and A328F mutant. With A328F BChE mutant, intrinsic K’ value ( ” [I]20.5 extrapolated to [S] = 0) for NB was 0.11 ± 0.02 lM. Compared to wild type BChE (K’ = 0.009 ± 0.002 lM), NB was found to be 12-fold less effective inhibitor of A328F mutant. These results suggest that alanine at position 328 in human BChE may be important in NB binding to BChE. Acknowledgement: This study was supported by a grant (SBAG-3677) from Scientific and Technical Research Council of Turkey. Keywords: Butyrylcholinesterase; nile blue; butyrylcholinesterase inhibition.

YSF.8 Features of characterization and structure of the biofilm matrix on the stainless steel surface M. Boretska, A. Ostapchuk and I. Kozlova Zabolotny Institution of Microbiology and Virology Microbial populations on the solid surfaces are capable to form highly organized structures – biofilms. A major role in this structures plays exopolymeric matrix – the polymer in which cells are immersed. Studying of the biofilms formed by corrosion dangerous sulphur cycle bacteria is needed to develop protection ways of large-scale metal structures as the primary site of microbial corrosion influence. The focus was to investigate the composition of exopolymeric complex and structural features of biofilms formed by acidophobic thiobacteria Thiobacillus thioparus 2M on the mild steel surface. The Albersheim method to determine monosaccharide composition (Agilent 6890N/5973 inert), the confocal laser scanning microscopy to structure visualization (LSM Pascal 5; Carl Zeiss, Germany) were used. Exopolymer’s monosaccharide composition in thiobacteria biofilms monoculture and their association with heterotrophic satellite differed from that one in plankton. In association biofilm’s matrix noted the most variety of monosaccharide composition. The xylose was typical only for biofilm growth both mono-and associative culture. The rhamnose, xylose, ribose and galactose were only found in biofilms associated cultures. Predominant monosaccharide as for mono-and for associatively biofilms was glucose. The complication of the species biofilms composition increases the impact of microbial corrosion on the metal previous work showed. The monosaccharide matrix composition depended on the species component of the biofilms. Therefore, the localization of carbohydrate in the structure were examined. The carbohydrate part of the bioflms matrix visualized by ConA + FITC (Sigma), specific for glucose, for cells visualization – DAPI (Sigma). Biofilms formed by the associative culture in an average of 40 microns thick, the surface is more uniform in comparison with monoculture biofilms were shown. The top layers of cells localized in the bottom carbohydrate layers. The lower layer of carbohydrates partially contained cells. This distribution was characteristic of both mono and for associative cul-

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YSF – Young Scientist Forum tures. Obviously thionic bacteria exocarbohydrate matrix played an important role in creating conditions for the attachment of cells T. thioparus 2 M, biofilm architectonics formation, as well as enhance the corrosion activity of the thiobacteria as shown.

YSF.9 Structural and functional study of ComE, a key actor in Streptococcus pneumoniae competence M. Boudes, D. Durand, M. Graille, A. Doizy, H. Van Tilbeurgh and S. Quevillon-Cheruel IBBMC, Universite´ Paris-Sud, Orsay, France Streptococcus pneumoniae is the leading cause of communityacquired infections. Its global success might in part be explained by its genetic transformation, which consists of the internalization and the incorporation in its chromosome of exogenous DNA. Transformation is turned on in cells which are in a physiological state called competence. Its induction depends on the two-component system ComD-ComE. The response regulator (RR) ComE is phosphorylated by the histidine-kinase ComD, and acts as transcriptional activator of competence-specific operons. These genes encode in proteins involved in processing and recombination of exogenous DNA, but also in the virulence of S. pneumoniae. The prototypical RR contains a conserved regulatory domain, linked to a variable effector domain. The majority of RRs (63%) contain DNA binding effector domains. Among them, 5% interact with DNA through the recently characterized LytTR domain. They form the AgrA/LytR family and regulate production of many important virulence factors. Despite their interest for drug development, no full-length RR structure has been determined yet within this family. ComE belongs to the AgrA/LytR family. We initiated a structural study of the ComE non-phosphorylable D58A mutant, and solved its X-ray 3D structure at 3.4A resolution. As expected, ComE D58A contains a regulatory domain and a LytTR domain. The two domains are linked by a long non-structured linker. Interestingly, ComE D58A forms a dimer in the crystal. The regulatory domains are linked by a two-fold symmetry axis within the dimer, whereas the LytTR domains are related by both a translation and rotation (head–to–tail). This asymmetric dimer configuration is facilitated by the flexibility of the linker. The sequence of the ComE promoter is known and consists of two direct repeats. Although ComE D58A was crystallized without DNA, the dimer conformation seems to be consistent with promoter binding, which incited us to set out for a functional mechanistic study.

YSF.10 Genotoxicity associated with oxidative damage in liver and kidney of mice exposed to dimethoate subchronic intoxication I. Boussama-Ayed, K. Rjiba, A. Moussa, N. Mnasri and H. Bacha Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Monastir, Tunisia Because of the widespread use of pesticides for domestic and industrial applications, the evaluation of their toxic effects is of major concern to public health. The aim of the present study was to investigate the propensity of Dimethoate (DM), an organophosphorous pesticide, to cause oxidative damage in liver and kidney of mice and its associated genotoxic effect. DM was administered intraperitoneally at doses of 1, 5, 10, 15 and 30 mg/kg body weight for 30 consecutive days in balb/c mice.


YSF – Young Scientist Forum Oxidative stress was monitored in kidney and liver by measuring the malondialdehyde level, the protein carbonyls generation and the catalase activity. Our results indicated an increase in lipid peroxidation and protein carbonyl levels in liver and kidney in a dose dependant manner. The catalase activity was found to be significantly increased in liver and kidney since the dose of 5 mg/Kg body weight. Genotoxicity of DM was assessed by the comet assay, the chromosome aberration assay and micronucleus test. Our study demonstrated that DM induced DNA damage in liver and kidney of treated mice in a dose dependant manner, There was a significant increase (p < 0.05) in the frequency of micronucleated cells following DM administration. Chromosome aberration assay revealed also a significant increase in percentage of chromosome abnormalities in a dose dependent manner. These results demonstrated that DM was genotoxic and this genotoxicity was associated to DM-induced oxidative stress.

YSF.11 The overexpression of HER family members modulates the efficacy of EGFR inhibitors in prostate cancer M. D. Carrion-Salip, R. de Llorens and A. Massaguer Biology Department, Faculty of Science, University of Girona, Girona, Spain The deregulation of the Human Epidermal Growth Factor Receptors (EGFR) pathway plays a major role in the pathogenesis, progression and metastasis of prostate cancer (PCa). However, the clinical effectiveness of therapies targenting EGFR in PCa is limited. The ability of prostate cells to upregulate the expression of alternative receptors of the EGFR family (HER2, HER3 and HER4) as well as the EGFR family ligands might represent an important resistance mechanism to maintain the cellular proliferation after EGFR blockage. In this work, we have analyzed the expression of the EGFR/ HER receptors and eight ligands of the EGFR family in two androgen-independent human prostate carcinoma cell lines (DU145 and PC3) prior and after their treatment with three EGFR-inhibitors (Cetuximab, Gefinitib and Erlotinib). Our results revealed that EGFR-blockage shortly induced an enhanced gene expression of three ligands (EGF, Betacellulin and Neuregulin) along with the HER4 receptor in the DU145 cells. Interestingly, the alternative binding affinities of these ligands might activate all possible HER heterodimers to compensate the loss of EGFR function. In contrast, slight differences were observed in the PC3 cell line. PC3 cells lack the phosphate and tensin homolog tumor (PTEN) gene, which leads to a continuous downstream activation of EGFR in a ligand-independent manner. The sensitivity of PC3 cells to EGFR inhibitors, determined as IC50 values and the inhibition of the cell growth, was markedly lower compared to DU145 cells. We have also examined the expression of EGFR family members in a Erlotinib-resistant cell line (DUErR) established after 6 months of DU145 cells exposure to Erlotinib. The DUErR cells presented a significant increase in HER2 and HER3 mRNA and protein levels along with higher mRNA levels of TGFa and Neuregulin, a cognate ligand of HER3. In accordance, the phosporylation of HER3 was markedly increased in the DUErR cells. Thus, EGFR blockage might be circumvented by the alternative signalling through HER3 in the resistant cells. In conclusion, our results indicate that the ability of androgenindependent PCa cells to regulate the autocrine expression of alternative EGFR receptors and ligands may represent a relevant molecular mechanism of resistance to drugs targeting EGFR.

Abstracts YSF.12 Mapping of the cell binding site in an extracellular matrix protein S. Carulli, K. Beck, H. Lortat-Jacob, F. Letourneur and P. Rousselle IFR128 BioSciences Gerland-Lyon Sud, Institut de Biologie et Chimie des Proteínes, FRE 3310, CNRS, Univ. Lyon1, 7 passage du Vercors, Lyon, France The heparan sulfate proteoglycan receptor syndecan-1 (SYN1) interacts with the C-terminal domain of the extracellular matrix protein laminin-332 (LN332) to participate in keratinocyte migration by inducing formation of cytoskeleton related protrusive structures. We have shown that SYN1 mediated cell adhesion occurs in heparan sulfate and chondroitin sulfate dependent manner and that these two glycosaminoglycan (GAG) chains bind independently to this particular domain with different affinities. To identify residues involved in the interaction of SYN1 with LN and to apprehend the molecular basis of its GAGs interaction specificity, we have used a site-directed mutagenesis approach of the recombinant LN fragment. The residues identified as conserved heparin binding residues throughout LNs, as well as ‘‘candidate’’ basic residues identified through predictive approaches, have been replaced by the neutral residue glutamine. All proteins carrying a hexa-histidine tag were expressed in mammalian cells, purified and characterized biochemically. Circular dichroism studies showed that the overall structure of each mutant is comparable to that of the wild type protein. Heparin affinity chromatography analysis allowed us to identify a major heparin binding site (HBS) in the LN domain surrounded by several minor GAG binding sites. Surface plasmon resonance analysis of all mutated proteins-Heparan sulfate interaction confirmed these results. These findings were well correlated with our in cellulo SYN1 mediated cell adhesion as the lack of this major HBS totally abrogated cell adhesion. Pull down experiments allowed us to show that this HBS sequence is responsible not only for the interaction of the receptors SYN1 but also for SYN4 suggesting that additional cellular functions may be carried by this sequence. Our structural predictions suggest that the C-terminal end of LN332 encompasses a major GAG binding site surrounded by a track of converging positively charged residues.

YSF.13 Biochemical characterization of Plasmodium falciparum CDPK4 and Anopheles gambiae TDO: two antimalarial targets A. Cavagnino, M. Rizzi and F. Rossi DiSCAFF, University of Piemonte Orientale, Novara, Italy Transmission of malaria requires an obligatory biological interplay between female Anopheles mosquitoes and a parasite of the genus Plasmodium, the etiological agent of the disease. The degradation of L-tryptophan by the mosquito is mandatorily initiated by A. gambiae 2,3-Tryptophan dioxygenase (AgTDO) and results in the production of Xanthurenic Acid (XA). The progression of Plasmodium life cycle in the invertebrate host takes place in the midgut and is triggered by XA. Plasmodium falciparum Calcium Dependent Protein Kinase IV (PfCDPK4) is recognized as a molecular switch that translates the XA-induced calcium signal into a cellular response by regulating cell cycle progression. CDPKs family is characterized by the presence of a regulatory domain in the C-terminal region that is able to activate the enzyme following calcium binding. We reported an advanced biochemical characterization that included electrochemical studies on AgTDO, providing new


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Abstracts insights into the mechanism sustaining catalysis. We performed an enzymatic characterization of PfCDPK4 using a non-radioactive method. Furthermore, by means of kinetic and structural analysis we identified PfCDPK4 phenothiazine-based inhibitors. These last data highlighted the CDPKs regulatory domain as a promising target for the development of innovative enzyme inhibitors. Overall, our results represent a basis for the development of small molecules interfering either with the mosquito or the parasite biology, to be used as insecticides or as malaria transmission blocking agents.

YSF.14 (S2.1.5) Role of microRNAs in Duchenne muscular dystrophy and in muscle differentiation M. Cesana, D. Cacchiarelli, J. Martone, E. Girardi, T. Incitti, M. Morlando, C. Nicoletti, T. Santini, O. Sthandier, L. Barberi, A. Auricchio, A. Musaro` and I. Bozzoni Department of Biology and Biotechnology ‘‘C. Darwin’’, Institut Pasteur Cenci-Bolognetti and IBPM – Sapienza, University of Rome, Rome, Italy Duchenne Muscular Dystrophy (DMD), caused by mutations in the dystrophin gene, is one of the most severe myopathies. Among different therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional mRNA. Making use of exon skipping strategy we demonstrated that in DMD, the absence of dystrophin at the sarcolemma delocalizes and downregulates Nitric Oxide Synthase (nNOS); this alters HDAC2 S-nitrosylation and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes crucial in DMD physiopathology. Namely, we identified miR-1 as regulator of the redox state of the cell through modulation of the G6PD enzyme while miR-29 controls the fibrotic process targeting extracellular matrix proteins. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being expressed in activated satellite cells before dystrophin expression. In these cells, miR-206 contributes to muscle regeneration through repression of the satellite specific factor Pax7. We conclude that: (1). The pathway activated by dystrophin/ nNOS controls key miRNA circuitries increasing the robustness of the muscle differentiation programme. (2). Specific miRNAs are induced during muscle regeneration controlling the timing of mRNA expression during myoblasts differentiation.

YSF.15 (S3.2.6) The signal peptides and the early mature domain cooperate for efficient secretion

YSF – Young Scientist Forum energy and activating the translocase. The activation of the translocase machine and the trapping of the preprotein in the channel, assures the multiple catalytic cycles and finally the secretion. In order to study the effect of the signal peptide at secretion, 22 different signal peptides were fused in front of the PhoA (Alkaline phosphatase) gene and the in vivo and in vitro secretion efficiency of the enzymes was measured. The preproteins had remarkable differences at their secretion efficiencies and were classified as inefficient, efficient and very efficient secretors. The inefficient secretors could not lower the activation energy of the translocase. Although the lowering of the activation energy is a signal peptide driven process, a preprotein with a sec signal peptide could not lower the activation energy. The inefficiency was not due to a dysfunctional signal peptide but because of the incompatibility between the signal peptide and the mature domain that follows. We conclude that the signal peptides and the early mature domain cooperate for efficient secretion.

YSF.16 (S2.2.5) Characterization of new small RNA populations in mouse embryonic stem cells C. Ciaudo1,2, J. Toedling3, I. Okamoto2, N. Servant3, E. Barillot3, E. Heard2 and O. Voinnet1 1 Swiss Federal Institute of Technology (ETH-Z), Zurich, Switzerland, 2Institut Curie, CNRS UMR3215, rue d’Ulm, Paris, France, 3Institut Curie, Service Bioinformatique, Paris, France A basal network of gene regulation orchestrates the processes ensuring maintenance of cellular identity and genome integrity. Small RNAs generated by the ubiquitous RNAse-III Dicer have recently emerged as central players in this network, by mediating gene silencing at the transcriptional or post-transcriptional level via RNA interference (RNAi). To gain insight into their potential developmental functions in mammals, we have characterized small RNA expression profiles during Embryonic Stem (ES) cell differentiation as ES cells provide an invaluable model for early mammalian development. Extensive used of new deep sequencing approaches enable us to better characterize microRNA populations but highlight important discrepancies between different technologies (Roche, Illumina and Life Sciences-ABI). This work has also uncovered the existence of novel small RNA entities that accumulate during differentiation of mouse ES cells, and that map to both repetitive and unique regions of the genome, including genes. Some of these small RNAs have characteristics of cognate siRNA populations while others display strand bias and length distributions that evoke their biogenesis through RNA surveillance pathways, in a dicer-independent manner. These observations demonstrate the power of ES cells in defining the repertoire of small RNAs and their dynamics in mammals. They also underline the potential that ES cells provide for defining the biogenesis and the functional roles of small RNAs in mammals, where studies in early embryos are challenging.

YSF.17 Temperature induced reactive oxygen species (ROS) in rat liver mitochondria

K. I. Chatzi1,2, G. Gouridis1,2, G. Orfanoudaki1,2, M. Koukaki2, I. Tsamardinos3, S. Karamanou2 and A. Economou1,2 1 Department of Biology, University of Crete, Crete, Greece, 2 Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology-Hellas, Crete, Greece, 3Institute of Computer Science, Foundation of Research and Technology-Hellas, Crete, Greece

R. Dakarviene¨, L. Degutyte¨-Fomins, R. ‡uˆkiene¨, V. Mildaþiene¨ and Z. Naueìene¨ Department of Biochemistry and Biotechnologies, Vytautas Magnus University, Centre of Environmental Research

One third of the bacterial proteins become secreted or membrane inserted. The signal peptide is necessary but not sufficient for the export of the preproteins from the cytoplasm. During secretion the signal peptide is responsible for lowering the activation

Mitochondria (Mt) are highly dynamic organelles that exhibit morphological and biochemical changes during physiological cell metabolism and stress responses. Age-related diseases, including cancer, type 2 diabetes, Alzheimer, Parkinson diseases, show

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YSF – Young Scientist Forum clear correlation with Mt dysfunction and reactive oxygen species (ROS). We have focused our study on response of Mt from normal tissues to hyperthermia that might affect healthy tissues when neighbouring tumours are treated by thermoablation or explosion to mild hyperthermia. We investigated the generation of ROS in isolated liver Mt derived from 1 to 7 months Wistar rats, both genders at 37–45C temperature range. Internal ROS generation was determined fluorimetricaly with dihydrodichlorofluorescein diacetate (H2DCFDA) and external – with Amplex Red. The results showed that at 37C much less ROS are generated by female rat Mt, as compared to male rat Mt. The difference for internal ROS generation was much larger (30% less in female than in male Mt) than that for external ROS production (about 10%), though statistically significant difference was found for 2 month old rats only. Rising temperature above normal, the amount of external ROS increased but the age and gender determined differences became smaller. Temperature induced external Mt ROS generation for young rats was less 8% and 12% for male and female at 40C (compared to 37C) and 14% and 18% at 45C, respectively; than 6–7 month rats ROS generation at 40C was higher 14 ir 17% for male and female and 26% and 34% at 45C respectively. At 45C the amount of internal ROS was 22% less in female Mt. The highest response to hyperthermic stress was found in 2 month female rat Mt at 45C temperature – internal ROS content at 45C was higher by 1.6 times comparing to that at 37C.

YSF.18 Functional analysis of the NF-jB signaling pathway protein TANK A. Dalmizrak1, F. Renner2 and M. L. Schmitz2 1 Department of Medical Biology, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey, 2Institute of Biochemistry, Medical Faculty, Justus-Liebig-University, Giessen, Germany NF-jBs are transcription factors that can be activated by cytokines, infection agents and DNA double chain breaks, resulting in the regulation of several genes implicated in immune response, inflammation, survival and cancer. Activation and inactivation of the NF-jB signaling pathway is dependent on the interaction between proteins acting in this pathway. TANK is one of these proteins with a role in the canonical pathway and capable of activating the NF-jB signaling pathway by interacting with IKKe. In purpose to determine the interaction between the TANK and IKKe proteins 10 serine and threonine amino acids which were thought to be phosphorylated by post-translational modifications in the TANK protein sequence, were mutated to alanine. A C-terminal deletion mutant (DC 358–425) of TANK was also cloned. As a result of these studies, phosphorylation of TANK by IKKe was observed more strongly in the wild type TANK compared to the mutant type. Depending on increasing IKKe concentrations, elevated phosphorylation levels of wild type and mutant TANK proteins were also detected. Furthermore, the protein which had the 10 amino acid changes and was also C-terminally truncated (DC95) again displayed a slight phosphorylation. To understand the relationship between the TANK and SUMO1 proteins, four lysine amino acids in the TANK protein sequence were mutated to arginine amino acids. After phosphorylation by IKKe, TANK was still found to interact with SUMO1. In recent studies, lysine residue at position 282 have been shown to play an important role in modification of TANK by SUMO. As a conclusion, the identification of post-translational modifications of the TANK protein will help us to further understand the role of this protein in the NF-jB signaling pathway and in the pathogenesis of diseases.

Abstracts YSF.19 Molecular modelling of dioxygen pathways inside the protein matrix: application to CotA laccase J. M. Damas and C. M. Soares Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa Many enzymes throughout the biological world catalyze reactions that involve dioxygen or other small gaseous molecules. The catalytic activity of those enzymes depends on the accessibility and affinity of the gaseous substrate to the catalytic centre, and, ultimately, its reactivity there. The structural determinants of these molecular events are most of the times inaccessible to experimental approaches, specially the diffusion of dioxygen, where X-ray crystallography with xenon probes is probably the best experimental approach. In this case, biomolecular modelling and simulation comes as a new and powerful tool to understand the molecular details behind dioxygen diffusion inside protein matrixes and complement experimental approaches on dioxygen biocatalysis. This information may be of utmost importance for biotechnological applications, where specific aminoacid mutations on a known enzyme may allow us to design a new enzyme with optimized rates of reaction. One example of such an enzyme may be CotA laccase, a model bacterial multicopper oxidase which catalyzes the oxidation of different substrates with the reduction of dioxygen to water inside the enzyme. We have studied diffusion of dioxygen inside CotA laccase through molecular dynamics. We have also performed a particle insertion technique, which allows us to draw the dioxygen affinity maps inside CotA laccase through the free energy estimation of the placement of a dioxygen molecule anywhere inside the protein. These approaches allow us to define and study the possible minimum energy paths towards the catalytic centre.

YSF.20 Characterization of antioxidant/antiinflammatory properties and apoA-I-containing subpopulations of HDL from family subjects with monogenic low HDL disorders G. Daniil1, A. A. P. Phedonos1, A. G. Holleboom2, M. M. Motazacker2, L. Argyri1, J. A. Kuivenhoven2 and A. Chroni1 1 Institute of Biology, National Center for Scientific Research ‘‘Demokritos’’, Athens, Greece, 2Department of Experimental Vascular Medicine, Academic Medical Centre, Amsterdam, the Netherlands Abstract Background: Genetic factors regulate both high-density lipoprotein (HDL) levels and functionality, thus affecting HDL antiatherogenic properties. We characterized the HDL antioxidant/ anti-inflammatory properties and apoA-I-containing subpopulations in families with monogenic HDL disorders. Methods: Subjects with mutations in apolipoprotein A-I (apoAI), ATP-binding cassette transporter A1 (ABCA1) or lecithin:cholesterol acyltransferase (LCAT) and family controls were studied. HDL antioxidant/anti-inflammatory properties were assayed by an in vitro fluorometric method and HDL paraoxonase-1 (PON1), platelet activating factor-acetylhydrolase (PAF-AH), LCAT, malondialdehyde (MDA), PAF and serum amyloid A (SAA) were measured. ApoA-I-containing HDL subpopulations were analyzed by two-dimensional nondenaturing gel electrophoresis. Results: ApoA-I heterozygotes and subjects with partial or complete ABCA1 or LCAT deficiency had HDL with reduced antiox-


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Abstracts idant/anti-inflammatory properties and increased MDA levels. HDL-PON1 activity was reduced in apoA-I heterozygotes and in subjects with complete ABCA1 deficiency. HDL-PAF-AH activity was reduced in subjects with partial or complete ABCA1 deficiency or complete LCAT deficiency. HDL-LCAT activity was reduced in subjects with partial or complete LCAT deficiency. HDL-PAF levels were increased in apoA-I heterozygotes. All subjects had similar HDL-SAA levels. ApoA-I, ABCA1 and LCAT heterozygotes were depleted of the large a1 HDL subpopulation. Some LCAT heterozygotes showed also reduction of the a2 subpopulation. Subjects with complete LCAT deficiency showed mostly the small a4 HDL subpopulation and with complete ABCA1 deficiency the a4 and preb HDL subpopulations. Conclusions: This study shows that mutations in apoA-I, ABCA1 and LCAT have direct effect on the antioxidant/antiinflammatory properties of HDL. Furthermore, our study shows the effect of specific mutations on the apoA-I-containing HDL subpopulations profiles.

YSF – Young Scientist Forum immunoprecipitation data evidenced about complex formation between LMP2A and ITSN1 in vivo in different cell types. SH3domains of ITSN1 were sufficient to precipitate LMP2A in vitro, thus it was supposed that ITSN1 binds. Interaction between LMP2A and ITSN1 was found to be a superposition of interaction of SH3-domains of ITSN1 with -PXXP- motives of LMP2A and ITAM-motives of LMP2A with SH2-domain of adaptor protein Shb that bind simultaneously ITSN1 and LMP2A. Shb was shown to be phosphorylated and activated in this complex. Previously, LMP2A was shown to function as a ubiquitin-trap, forcing some kinases to proteosome degradation. We have found that complexes consisting of ITSN1 and LMP2A containing ubiquitin-ligases AIP4 and c-Cbl target ITSN1 for degradation. Moreover it was shown that AIP4 mediates ubiquitynation of ITSN1. Current findings provide new data about LMP2A-driving signalosome assembly and functioning addressing question about coupling of its traffic and signaling activity.

YSF.21 Galectin-3 activates b1-integrins through CD98

YSF.23 Microexon-based regulation of ITSN1 and Src SH3 domains specificity in brain

G. Delgado, J. Fort and M. Palacı´ n Institute for Research in Biomedicine-Barcelona, Universitat de Barcelona, Spain

M. Dergai, O. Dergai, I. Skrypkina, L. Tsyba, I. Zlatskiy and A. Rynditch Institute of Molecular Biology and Genetics, NASU, Kiev, Ukraine

Galectin-3 is a member of the b-galactoside-binding proteins that interacts with glycosilated plasma membrane receptors triggering calcium signaling, b1,3-integrin activation thus modulating cell adhesion, spreading and motility. One of its known interacting proteins is CD98, the heavy subunit of six different heteromeric amino acid transporters. The molecular structure of CD98 was determined in our lab in 2007, and it is closely related with aamylases. CD98 constitutively associates with b1-integrins and plays an essential role in its activation: antibodies against CD98 block fibronectin-dependent integrin adhesion. We have demonstrated that galectin-3 activates AKT, thus leading to cell surviving, preventing anoikis and promoting anchorage independent cell growth, a crucial phenomenon in metastasis of cancer cells, trophoblast implantation and lymphocyte extravasation. We want to elucidate the mechanism by which galectin-3 activates integrin through CD98; using stem cell-derived fibroblasts knock out for CD98, recombinant proteins and fluorescent confocal microscopy.

The SH3 domains function as protein-protein interaction modules in assembly of signalling and endocytic protein complexes. Here we report about the mechanism of regulation of the binding properties of the SH3 domains in neurons by alternative splicing. Previously, we have shown that the structure and function of SH3A domain of ITSN1 is affected by neuron-specific inclusion of five amino acids. So far, only one other protein, called Src, was shown to undergo similar regulation. Sequence analysis of ITSN1 and Src genes revealed that their alternative microexons are conserved in vertebrates and not specific for invertebrates. We showed that neuron-specific ITSN1 transcripts containing the microexon 20 could be detected during the earliest stages of the nervous system development since the first neurons start to differentiate. Further, structure modelling made it possible to uncover possible mechanism of regulation of SH3A domain binding properties by microexon 20. The mechanism was suggested to rely on changes in position of charged amino acids within interaction interface. Using mutational analysis we confirmed that this is realized to modulate ITSN1 (SH3A domain) binding to neuron-specific GTPase dynamin 1. Similar microexon-based mechanism was revealed for regulation of Src SH3 domain binding to dynamin 1. The microexon-encoded insert contain residue that abolishes interaction of the SH3 domain with dynamin 1. Thus, obtained data evidences that microexons provide a mechanism for the control of brain-specific interactions of ITSN1 and Src with protein partners.

YSF.22 Latent membrane protein 2A (LMP2A) of Epstein-Barr virus interacts with intersectin 1 and targets it for degradation O. Dergai, M. Dergai, I. Skrypkina, L. Matskova, L. Tsyba, G. Winberg and A. Rynditch Institute of Molecular Biology and Genetics, NASU, Kiev, Ukraine Epstein-Barr virus is a member of the herpesvirus family and one of the most common human viruses. EBV is associated with a number of human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma and the epithelial cell malignancy nasopharyngeal carcinoma (NPC). Only restricted set of viral genes is expressed within latent phase: LMP1, LMP2A, LMP2B, EBNAs and EBERs. Latent membrane proteins are key player of infected cells transformation. But little is known about mechanism governing internalization and trafficking through cells compartment. The aim of current work is to identify protein-protein interaction that allows latent membrane proteins to get access to the host endocytic machinery. Here we report about interaction between viral protein LMP2A and endocytic adaptor intersectin 1. Our

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YSF.24 Transcriptional regulation of a-synuclein G. Dermentzaki, R. L. Clough and L. Stefanis Division of Basic Neuroscience, Biomedical Research Foundation of the Academy of Athens a-Synuclein (SNCA) is an abundant neuronal protein linked to the development of neurodegenerative diseases, and in particular Parkinson’s disease (PD). Genetic overexpression or missense point mutations of SNCA lead to PD in humans, and its overexpression is sufficient to cause PD in some animal models. We previously identified elements in the 1st intron of SNCA that are important in its transcriptional regulation in PC12 cells in


YSF – Young Scientist Forum response to treatment with NGF and bFGF (Clough and Stefanis, 2007). Using a small-scale exonuclease deletion approach we aimed to identify the transcription factor (TF) binding sites in intron 1 responsible for this induction. Multi-species sequence alignment and promoter analysis identified two putative binding sites for TFs ZSCAN21 and HNF4. We further characterized the role of ZSCAN21 in the regulation of SNCA. A luciferase construct lacking the ZSCAN21 binding site exhibited a 40% reduction in activity when compared to control sequence. Electromobility shift assay with PC12 nuclear extract identified a specific shift for a biotin-labeled ZSCAN21 probe. RT-PCR verified the expression of ZSCAN21 in naı¨ ve and NGF-treated PC12 cells, as well as in different areas of the rat brain, including the ventral midbrain. siRNA against ZSCAN21 reduced the activity in the luciferase assay to the same levels as for constructs lacking this sequence, and significantly inhibited the protein expression of SNCA in PC12 cells and cortical neurons, thus establishing a role for ZSCAN21 in the transcriptional control of SNCA in these model systems. In order to extend these results in vivo, we intend to use stereotactic injections of lentivirus expressing shRNA against ZSCAN21 in the rat substantia nigra. Such studies may cement ZSCAN21 as an important regulator of SNCA transcription, and may provide potential therapeutic targets not only for PD but also for other synucleinopathies.

YSF.25 (S11.2.5) A genome-scale protein interaction profile of Drosophila p53 uncovers additional nodes of the human p53 network G. Di Minin1,2*, A. Lunardi1,2*, P. Provero3, M. Dal Ferro1,2, M. Carotti1,2, G. Del Sal1,2 and L. Collavin1,2 1 Laboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie (LNCIB), Area Science Park, Trieste, Italy, 2 Dipartimento di Scienze della Vita, Universita` degli Studi di Trieste, Trieste, Italy, 3Molecular Biotechnology Center and Dipartimento di Genetica, Biologia e Biochimica, Universita` degli Studi di Torino, Torino, Italy *A .L. and G .D. M. contributed equally to this work. The genome of the fruitfly Drosophila melanogaster contains a single p53-like protein, phylogenetically related to the ancestor of the mammalian p53 family of tumor suppressors. We reasoned that a comprehensive map of the protein interaction profile of Drosophila p53 (Dmp53) might help identify conserved interactions of the entire p53 family in man. Using a genome-scale in vitro expression cloning approach, we identified 91 previously unreported Dmp53 interactors, considerably expanding the current Drosophila p53 interactome. Looking for evolutionary conservation of these interactions, we tested 41 mammalian orthologs and found that 37 bound to one or more p53-family members when overexpressed in human cells. An RNAi-based functional assay for modulation of the p53 pathway returned five positive hits, validating the biological relevance of these interactions. One p53 interactor is GTPBP4, a nucleolar protein involved in 60S ribosome biogenesis. We demonstrate that GTPBP4 knockdown induces p53 accumulation and activation in the absence of nucleolar disruption. In breast tumors with wildtype p53, increased expression of GTPBP4 correlates with reduced patient survival, emphasizing a potential relevance of this regulatory axis in cancer.

Abstracts YSF.26 Mitotic entry regulation by Golgi complex partitioning: a novel mitotic checkpoint R. I. Cervigni1,2, M. L. Barretta1,2, D. Corda1 and A. Colanzi1,2 1 Institute of Protein Biochemistry, National Research Council (IBP-CNR), Naples, Italy, 2Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy The Golgi apparatus is a continuous membranous system, known as the ‘‘Golgi ribbon’’, which is maintained in the pericentriolar region of cells. One peculiar aspect of Golgi biogenesis is the mechanism of its mitotic inheritance, which involves the progressive and reversible disassembly of the ribbon structure into dispersed elements through a multistage process that starts during the G2 phase of cell cycle. This fragmentation must be precisely regulated for the optimal distribution of a functional Golgi complex to each of the daughter cells. Importantly, the first step of the fragmentation process, the severing during G2, is necessary for entry into mitosis, thus defining the Golgi checkpoint. We have recently demonstrated that the G2-specific Golgi fragmentation stage is concomitant with centrosome recruitment and activation of the mitotic kinase Aurora-A, an essential regulator for entry into mitosis. Using a microinjection-based experimental approach, we showed that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell-cycle progression. Overexpression of Aurora-A overrides this cell-cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. Overall, our findings reveal the existence of novel mechanisms that upon a block of Golgi fragmentation, lead to inhibition of the recruitment of Aurora-A to the centrosome during early G2, by acting either directly on Aurora-A or indirectly on an AuroraA activator. To investigate the role of possible candidates we developed a series of new experimetal approaches that are based on: (i) an ‘‘acute’’ transfection assay to reproduce the acute block of Golgi fragmentation, (ii) the inducible expression of proteins that act as ‘‘blockers’’ of Golgi fragmentation and (iii) fully automated image acquisition. The preliminary data are discussed.

YSF.27 Selection and characterisation of cyclic peptide inhibitors of the AcrAB-TolC multidrug efflux pump M. Doumit and P. Soumillion Laboratoire de Biochimie et Geńe´tique Molećulaire Bacte´rienne, Institute of life sciences, Catholic University of Louvain (UCL), Louvain-la-Neuve, Belgium A way to address the problem of multi resistance in infectious diseases is to develop strategies for identifying combinations of antibiotics with non-toxic inhibitors that can restore the activity of an antibiotic against which resistance has developed. In this project, we are searching for inhibitors of multidrug efflux pumps of gram-negative bacteria such as AcrAB-TolC, a well characterized model pump of Escherichia coli. Starting from combinatorial libraries of small cyclic peptides biosynthesized in E. coli,1 a selection strategy was designed for identifying peptides that are sensitizing the bacteria to an antibiotic compound because they block the efflux pump: upon addition of a bacteriolytic antibiotic such as oxacillin, sensitized bacteria are lysed and the plasmids encoding the peptides of interest are released into the supernatant, selectively recovered by centrifugation and re-introduced in new bacteria by transformation. This selection is qualified as anti-Darwinian because the population evolves towards an increase in antibiotic sensitivity.


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Abstracts A model selection was initially optimized. Six rounds of selection were then performed on collections of hexa-, hepta- and octa- cyclic peptides with cytoplasmic and periplasmic localization. Sensitized clones have been selected and are currently being characterized.

YSF.28 The bZIP transcription factor ATF3 is a novel mediator of the renin- angiotensin system in the heart O. Elhanani and A. Aronheim Department of Molecular Genetics, The Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa, Israel Angiotensin II (Ang II) is the primary effector molecule of the renin- angiotensin system. Ang II affects the function of many organs including the heart. Acute stimulation with Ang II regulates salt/water homeostasis, vasoconstriction and modulates blood pressure, while chronic exposure to Ang II plays a vital role in cardiac hypertrophy and remodeling. In our lab, we have discovered that acute administration of Ang II to mice, causes induction of the activating transcription factor 3 (ATF3) in the heart. ATF3 is an immediate early transcription factor that responds to many extracellular signals. In animal models, it has been shown that myocardial ischemia induces ATF3 in the heart. It has also been shown that transgenic mice with cardiac specific expression of ATF3 display bi-atrial enlargement, myocyte degeneration and fibrosis. In addition, the transgenic hearts exhibited reduced contractility. Interestingly, while a and b adrenergic agonists- Phenilephrine and Isoproterenol cause ATF3 induction in all four chambers of the mouse heart, Ang II dependent induction of ATF3 occurs specifically in the left side of the heart. ATF3 induction represents a cell autonomous process that is independent of the increase in blood pressure. We further characterized Ang II dependent signaling pathways from membrane bound receptor to its nuclear target ATF3. We show that ATF3 induction in the heart requires both the Angiotensin type 1 and type 2 receptor subtypes as well as EGFR transactivation and the AKT pathway all of which have been linked to cardiac hypertrophy. Chronic Ang II exposure, suggests that ATF3 KO mice display reduced hypertrophy comparing with Wt mice. Collectively, we propose that ATF3 may have a key role in cardiac function and pathology.

YSF.29 (S16.1.5) Specific ER aminopeptidase 1 SNPs affect antigen processing in vitro and demonstrate substrate inhibition kinetics I. Evnouchidou1, R. Kamal2, S. S. Seregin2, Y. Goto3, M. Tsujimoto3, A. Hattori4, P. V. Voulgari5, A. A. Drosos5, A. Amalfitano2, I. A. York2 and E. Stratikos1 1 Protein Chemistry Laboratory, IRRP, National Centre for Scientific Research ‘‘Demokritos’’, Agia Paraskevi, Greece, 2 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA, 3Faculty of Pharmaceutical Sciences, Teikyo-Heisei University, Ichihara, Chiba, Japan, 4Graduate School of Pharmaceutical Sciences, Kyoto University, Department of System Chemotherapy and Molecular Sciences, Sakyo, Kyoto, Japan, 5Rheumatology Clinic, Department of Internal Medicine, Medical School, University of Ioannina, Ioannina, Greece

YSF – Young Scientist Forum tide repertoire. ERAP1 has unusual enzymatic properties that fit well with its role in antigenic peptide processing. It trims more efficiently longer peptides compared to smaller ones and it demonstrates specificity for the whole sequence of its substrates and not only the N-terminus. Recently, coding ERAP1 single nucleotide polymorphisms (SNPs) have been associated with an autoimmune disease, ankylosing spondylitis (AS), suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated antigen processing. To investigate this possibility, we produced three ERAP1 allelic variants that have been associated with AS and analyzed their ability to process antigenic peptide precursors in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects both the Km and kcat of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations demonstrate substrate inhibition kinetics, a phenomenon not described before for this enzyme. Cell-based antigen presentation analysis is consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect antigen processing in vivo. Two recently solved crystallographic structures of ERAP1 allow the accurate mapping of these polymorphisms and provide hints towards understanding the structural basis for these effects. Furthermore, ERAP1’s elongated peptide binding cavity provides a structural basis that fits well with the observed substrate inhibition kinetics.

YSF.30 Targeting insulin receptor substrates for destruction as a therapeutic modality for cancers E. Flashner1,2, H. Reuveni1,2, K. Makedonski1,2, A. Shir1,2 and A. Levitzki1,2 1 Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel, 2NovoTyr Therapeutics, Tel Hai, Israel Insulin-like growth factor 1 receptor (IGF1R) and its signaling pathway play key roles in many human malignancies. This is why a number of IGF1R tyrosine phosphorylation inhibitors (tyrphostins) and antibodies are being developed as anti-tumor agents. IGF1R signaling is almost exclusively mediated by insulin receptor substrate (IRS) proteins – IRS1 and IRS2.Here we report, for the first time, on a unique family of tyrphostins that, in addition to being novel allosteric IGF1R kinase inhibitors, also lead to inhibitory serine phosphorylation and elimination of the IRS1/2 proteins. This attribute leads to long lasting inhibition of IGF1Rsignal transduction and cell growth in a wide range of cancer cell types and potent in-vivo anti-tumor effects on human melanoma and ovarian cancer in nude mice. Mechanistic studies show that the phosphorylation of IRS1 and IRS2 and their subsequent degradation is mediated, at least partially, by the proteasome. Interestingly, the serine phosphorylation on the IRS proteins, induced by this unique family of inhibitors, does not depend on IGF1R stimulation by IGF-1. These findings establish a new paradigm of signal transduction therapy: the irreversible elimination of a signal transducer that is essential for the survival of cancer cells.

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic pep-

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YSF – Young Scientist Forum YSF.31 Endocannabinoids and decidualization: CB1 mediates apoptosis through the generation of ceramide and activation of caspase 9 B. M. Fonseca1,2, M. Almada1, B. Macedo2, N. Teixeira1,2 and G. C. da Silva1,2 1 Laborato´rio de Bioquı´mica, Departamento Cieˆncias Biolo´gicas, Faculdade de Farmaćia e, 2IBMC, Universidade do Porto In rodents, decidual cells differentiate and proliferate during early pregnancy in response to implanting blastocyst. Later, decidual cells undergo a cycle of regression, which occurs mainly by apoptosis. This is essential to support placental function and, consequently, conceptus growth/development. However, the exact mechanisms controlling decidual regression are not fully understood. The discovery of ‘‘endocannabinoids’’ (ECs), the endogenous cannabis-like compounds, highlighted a new ‘‘clan’’ of lipid mediators. Although ECs have been associated with various physiological processes, their involvement in reproduction is still very intriguing [1]. We have previously shown that ECs machinery operates on decidual cells and found that AEA, the main endocannabinoid, induced apoptosis in decidual cells through cannabinoid receptor 1 (CB1) [2]. Ceramide levels, a lipid second messenger, have been shown to mediate cannabinoid induced apoptosis in vitro and also in vivo [3]. In the present study we intend to investigate which pathways may be involved in the apoptotic process observed during decidual regression. In that way, we have quantified ceramide levels by HPLC-MS/MS after AEA treatment, using primary decidual cell cultures. Moreover, we also studied how cannabinoid receptors are coupled to the generation of ceramide and the involvement of caspase 9. We found that AEA (10 lM) induced a significant increase in ceramide levels and in caspase 9 activity, effect inhibited by the pre-treatment with the CB1 receptor antagonist. The results suggest that AEA- induced apoptosis of decidual cells could be mediated by ceramide and in that way it may impar normal pregnancy, effect dependent on the activation of CB1 receptor. References: 1. Maccarrone, M. Hum Reprod, 2009, 24(7): 1771. 2. Fonseca, B. M., et al. Endocrinology, 2010, 151 (8): 3965–74. 3. Giuliano, M., et al. Int J Mol Med, 2006. 17(5): 811–9.

YSF.32 A critical analysis of a mathematical model for mitotic exit P. Freire, P. K. Vinod and B. Novak Department of Biochemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK Mitosis is the fundamental physiological process by which cells accomplish nuclear division after having successfully replicated their DNA content. This process is initiated by an increase in Cdk1 activity, which is followed by activation of cohesion cleaving separase and finishes when Cdk1 activity is switched off. In budding yeast, Cdc14 is a core phosphatase that counterbalances kinase activity at the end of mitosis. During most of the cell cycle, Cdc14 is sequestered in the nucleolus and kept inactive. Its activation occurs during anaphase and late mitosis and is regulated by two networks called FEAR (Cdc Fourteen Early Anaphase Release) and MEN (Mitotic Exit Network). In this work, we demonstrate how mitotic exit is orchestrated by Cdc20/APC, which functions as a critical node controlling both the cyclin-dependent kinase (Cdk1) and phosphatase (Cdc14) branches of the regulatory network. Further numerical analysis

Abstracts of the model shows the underlying consequences of keeping CycB-dependent Cdk1 activity at a high level. Firstly we analyzed mutant phenotypes in cells depleted of Cdc20. Secondly, we analyzed mutants expressing a stable version of Clb2 (Clb2-kd – lacking ken and destruction boxes). The results indicate a clear role of the positive feedback between Cdc14 and Cdc15 (MEN component) in Cdc14 release. Moreover, the model also unravels the main principles of recently discovered Cdc14 endocycles (Lu and Cross, 2009; Manzoni et al., 2010). In summary, this new budding yeast cell cycle model is a straightforward tool to comprehend the underlying governing principles behind the main events occurring in mitotic exit.

YSF.33 (S5.3.5) Assigning a role to the Dengue virus capsid protein during cellular infection J. M. Freire, A. S. Veiga1, N. C. Santos1, W. Kowalczyk2, D. Andreu2, A. T. Da Poian3 and M. Castanho1 1 Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Physical Biochemistry Unit, 2Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Barcelona, Spain, 3Instituto de Bioquı´mica Me´dica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Dengue Virus (DV) causes about 20.000 deaths due to viral haemorrhagic fever pathology and infects 50–100 million people every year. No effective treatment is available and several aspects of the viral multiplication and infectivity remain unclear. The functionalities of the capsid protein (DVCP), for instance, remain elusive. Two peptides derived from two conserved domains of DVCP were studied in the presence of oligonucleotides and biological membrane models (Large Unilamellar Vesicles) using spectroscopic techniques (Dynamic Light Scattering – DLS, Zeta-Potential, Fluorescence Spectroscopy and Laser Confocal Microscopy). Peptide R and Peptide M, these including respectively the putative DVCP RNA- and membrane-binding domains, show affinity for model lipid bilayers and for membranes of Peripheral Blood Mononuclear Cells (PBMC). DLS and energy transfer (FRET) experiments of both peptides with oligonucleotides revealed peptide/oligonucleotides complex formation with high affinity binding constants, which suggest the lack of a specific viral genome binding domain. These complexes also interact with PBMCs and with lipid model membranes (quantified by a novel Nernst partition model for supramolecular complexes). The biological implications of these results will be discussed.

YSF.34 NMR structural studies of a double transmembrane domain of the translocator protein C. Galvagnion, P. Montaville, Y.-M. Coı¨ c and N. Jamin CEA/iBiTecS/SB2SM and URA CNRS 2096 The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a small transmembrane (18 kDa) protein mainly involved in the translocation of cholesterol into the mitochondria, the rate limiting step of the steroids biosynthesis. TSPO is proposed to fold as a five a helix bundle. Mutagenesis and molecular modelling studies of mTSPO proposed that the cholesterol binding site of TSPO is located at the C-terminal of its fifth helix and highlighted the essential role of aromatic residues for the binding of cholesterol, such as Y152 and Y153. To date, no atomic detailed model of mammalian TSPO is avail-


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Abstracts able. Such model is, however, essential to characterise the structure-function relationships of the protein at the atomic level. Here, we present the structural studies of the helix-loop-helix domain of TSPO (TM45) encompassing the fourth (TM4) and fifth (TM5) a helices of the protein. In particular, the secondary and tertiary structures of the domain was characterised in different detergents using circular dichroism and solution nuclear magnetic resonance spectroscopy. The backbone resonances were assigned and sequential and medium range NOEs highlighted. Those preliminary results allow us to estimate the length and stability of the secondary structure elements: TM45 consists of two a helices TM4 and TM5, of 25 and 22 residues long (Q104-R128 and A133-V154), respectively, connected by a four residues loop. Long range NOEs are currently gathered and a structural model is under process.

YSF.35 Connexin43 inhibits the oncogenic activity of c-Src in C6 glioma cells E. Gangoso1, S. Herrero-Gonza´lez1, C. Giaume2, C. Naus3, J. M Medina1 and A. Tabernero1 1 Departamento de Bioquı´mica y Biologıá Molecular, Instituto de Neurociencias de Castilla y Leoń (INCYL), Universidad de Salamanca, Spain, 2INSERM U840, Colle`ge de France, Paris, France, 3Department of Cellular and Physiological Sciences, The Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada One of the characteristics of gliomas is a decrease in the expression of connexin43, a protein that forms gap junctions. Restoring connexin43 expression in glioma cells reduces their exacerbated rate of cell growth, although it is not yet known how connexin43 modifies the expression of genes involved in cell proliferation. Here, we show that restoring connexin43 to C6 glioma cells impedes their progression from G0/G1 to the S phase of the cell cycle by reducing retinoblastoma phosphorylation and cyclin E expression through the upregulation of p21 and p27. Interestingly, connexin43 diminishes the oncogenic activity of c-Src exhibited by glioma cells. By studying a Tyr247 and Tyr265 mutant connexin43, we show that these residues are required for connexin43 to inhibit c-Src activity and cell proliferation. In conclusion, by acting as a substrate of c-Src, connexin43 reduces its oncogenic activity and decreases the rate of glioma cell proliferation, potentially an early step in the antiproliferative effects of connexin43. Although c-Src is known to phosphorylate connexin43, this study provides the first evidence that connexin43 can also inhibit c-Src activity.

YSF.36 Ras dependent LKB1 hyperactivation controls proliferation in HCC tumor cells derived from GNMT deficient mice J. L. Garcı´ a-Rodrı´ guez, N. Martı´ nez-Lo´pez, M. Varela-Rey, V. Gutie´rrez-de Juan, Z. Luka, D. F. Calvisi, S. C. Lu, J. M. Mato and M. L. Martı´ nez-Chantar CIC bioGUNE, Centro de Investigacioń Biome´dica en Red de Enfermedades Hepa´ticas y Digestivas (Ciberehd), Technoology Park of Bizkaia, Bizkaia, Spain,Division of Gastrointestinal and Liver DIseases, USC Research Center for Liver Diseases, Southern California Research Center of Alcoholic Liver and Pacreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA HCC is a heterogeneous pathology with a variety of risk factors. Steatosis and NASH are hallmarks of non-alcoholic fatty liver disease (NAFLD). The molecular mechanisms of NASH to HCC

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YSF – Young Scientist Forum progression remain unknown. Human patients with liver cirrhosis at a high risk of HCC have impaired methionine metabolism and abnormal S-Adenosylmethionine(SAMe) levels. SAMe, the main methyl donor in cells plays a critical role in cell proliferation, differentiation and apoptosis. The two main enzymes involved in SAMe synthesis and catabolism are MAT(methionine adenosyltransferase) and GNMT(glycine N-methyltransferase) respectively. MAT1A and GNMT deficiency have been reported in human HCC. Our laboratory generated MAT1A-KO and GNMT-KO mice, with decreased and increased hepatic SAMe content, respectively. Both models develop HCC spontaneously. GNMT-KO mice develop steatosis that progresses to fibrosis and HCC, being DNA hypermethylation the main mechanism involved. We isolated a cell line derived from a hepatic tumour of GNMT-KO mice, OKER cells. Our data indicate that hyperphosphorylation of LKB1-Ser428 plays a critical role in proliferation of the tumour. SAMe excess promotes hyperactivation of Ras due to promoter methylation and subsequent silencing of RASSF1(Ras inhibitor). Wild type Ras pathway is responsible for the LKB1-hyperphosphorylation(Ser428) in an ERK/ P90RSK1-dependent fashion, leading to LKB1-AMPK misconnection. Treatment with demethylating agent, 5‘-azacytidine, abolished Ras pathway activation, induced AMPK activation and promoted apoptosis. Ablation of LKB1 triggered apoptosis. Finally, in a xenograft-model in nude mice, LKB1 suppression by IP injections of siRNA revealed decrease in tumour growth, increase of necrotic regions and AMPK activity emphasizing the proliferative role of LKB1 in OKER cells. Critically, our results open a novel therapeutical strategy in HCC treatment and highlight the importance of LKB1 activity so far considered as a tumor suppressor.

YSF.37 Novel features in Bacillus subtilis dUTPases J. Garcı´ a-Nafrı´ a1, M. Fogg1, M. Harkiolaki, R. Persson and K. S. Wilson1 1 York Structural Biology Laboratory, University of York, York, UK, 2Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford, UK, 3Semtech Metallurgy AB, Ideon Science Park, Lund, Sweden The presence of uracil in DNA can be either the result of cytosine deamination or of incorporation of dUMP by the promiscuous DNA polymerase. Excision repair mechanisms solve this problem by cleaving uracil and adding the right nucleotide. However high concentrations of uracil would produce massive incorporation into DNA and the extensive double strand breaks by the excision mechanisms produces double strand breaks and eventually cell death. To avoid this, cells produce dUTPase which hydrolyses dUTP into dUMP and PPi in an ion dependent manner. In addition, hydrolysis of dUTP aids in the production of dTTP whose precursor is dUMP. dUTPases are essential enzymes conserved from viruses and bacteria to higher organisms. The homotrimeric dUTPases are the most common and indeed best characterised forms. The human orthologue has been proposed as new chemotherapeutic target while the Plasmodium and Mycobacterium enzymes are potential drug targets for parasite annihilation. Bacillus subtilis has two dUTPases, a genomic (YncF) and a prophage form (YosS). For both we have determined crystals structures of the free enzymes and for dUTP analogue complexes. For the YosS enzyme we in addition have the structure of the enzyme complexed with both the product (dUMP) and an inhibitor dUDP. The structures show that the two Bacillus enzymes possess some key differences from all the others. Indeed they provide fresh insights into the mechanism as well as a novel feature: the


YSF – Young Scientist Forum position in the sequence of the otherwise strictly conserved PheLid residue is totally different to all other known dUTPases. This raises some questions regarding the proposed mechanism. .Preliminary crystals with compounds intended to mimic the transition state have recently been obtained, and their analysis is in progress.

YSF.38 The importance of breast cancer-endothelium communication in the expression of matrix molecules C. Gialeli1, M. Viola2, A. Passi2 and N. K. Karamanos1 1 Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece, 2Department of Biomedical and Experimental Sciences, Faculty of Medicine and Surgery, University of Insubria, Varese, Italy During development of carcinogenesis, tumor cells participate in several interactions with the tumor microenvironment as well as the surrounding cells (endothelial cells, fibroblasts). High level of hyaluronan (HA) is often associated with malignant progression in many cancers, such as breast cancer. Hyaluronan-rich matrices around tumors favor the cancer cells migration and infiltration of newly formed blood vessels. The aim of the present study was to determine the cellular responses between the breast cancer cell lines, MDA-MB-231 (highly metastatic) & MCF-7, and the human umbilical vein endothelial cells (HUVEC) using conditioned media (CM) and co-culture Transwell systems. We assayed that conditioned media from cancer and HUVEC cells decrease and induce the cell migration of HUVEC and cancer cells, respectively. Real-Time PCR analysis showed that the gene expression of CD44, HAS2, VCAM-1 and ICAM-1 in HUVEC is up-regulated, especially by CM from MDA-MB-231. In MDA-MB-231 and MCF-7, CD44 and HAS2 are up-regulated and down-regulated, respectively, by CM from HUVEC. A similar effect is demonstrated during co-culture of HUVEC and cancer cells. HA expression in culture medium of HUVEC cells is up-regulated by CM from cancer cells, in reference with CM added, whereas in cancer cells is not affected by CM from HUVEC. In co-culture system, HA expression is similar, highlighting the cell-cell communications. In conclusion, relatively little is known about the paracrine effects of tumor-endothelial cell interactions and it is of high interest to highlight key molecules participating in this cross-talk.

YSF.39 Impaired, glucocorticoid-independent, resolution of the inflammatory response in corticotropin releasing hormone (Crh)deficient mice following DSS- colitis P. Giannogonas, Z. Chaniotou, S. Theoharis, H. Pothoulakis and K. Karalis Biomedical Research Foundation of the Academy of Athens Inflammatory bowel disease (IBD) is a chronic, relapsing disease including ulcerative colitis and Crohn’s disease, with aetiology still non elucidated. It is currelntly accepted that it is dependent on complex interactions between genetic and environmental factors, with innate immunity and microbiota playing a critical role in its pathophysiology. In previous studies we have demonstrated the immunomodulatory effects of Corticotropin Releasing Hormone, or Factor, (CRH or CRF), a neuropeptide acting as the major mediator of the stress response, in intestinal inflammation

Abstracts in rodents. Using an experimental model of innate immunitydependent ulcerative colitis, the DSS model, we have recently shown that Crh deficiency is associated with increased susceptibility to disease development. To extend our understanding on the role of CRF in experimental IBD, we followed the course of DSS-colitis during the phase of tissue injury repair, and reversal of body weight loss in the Crh)/) mouse. To our surprise the disease was progressed in the Crh)/) mice 5 days following cessation of DSS treatment resulting in their severely compromised survival, while all wild-type mice recovered. The progressive disease in the Crh)/) mice was characterized by significant body weight loss, lack of regeneration of the inflamed epithelium, increased angiogenesis and fibrosis. These findings were not reversed by normalization of the glucocorticoid levels of the Crh)/) mouse, in further support of the direct role of Crh in this process. Our findings demonstrate the requirement of CRH for the intestinal epithelial repair in colitis and raise the hypothesis for a critical contribution of ‘‘a tissue stress response’’ in the resolution of inflammation and return to homeostasis.

YSF.40 Oxidative carbonylation study as a novel approach to identify damaged proteins with relevant functions in fermentation process R. Gomez-Pastor, P.-T. Roberto and M. Emilia Department of Biochemistry and Molecular Biology, University of Valencia, Spain, Department of Biotechnology, Institute of Agrochemistry and Food Technology, Spain Damages caused by ROS accumulation under adverse environmental conditions leads to oxidative alterations to different cell components. The formation and accumulation of protein-carbonyl by ROS seems to be the most important oxidative damage since it is a selective and an irreversible modification and may serve as a marker of oxidative stress, aging, and age-related diseases. Oxidatively modified proteins are generally dysfunctional, losing catalytic or structural integrity and increasing protein aggregates. The study of carbonylated proteins by 2D gel electrophoresis using 2,4-DNPH derivated samples and mass spectrometry identification has been used to identify damaged proteins along yeast biomass production where cells are exposed to severe growth conditions [1, 2, 3]. This approach allowed us to isolate the specific proteins that are affected by oxidative carbonylation with relevant interest in fermentation processes such as Adh1p, Pdc1p and several proteins related to heat shock proteins, ATP metabolism and TCA cycle. The obtained data can be directly correlated with the fermentative capacity detriment observed for S. cerevisiae after biomass propagation conditions [1, 2]. Besides, the oxidative stress depletion by TRX2 gene overexpression [2] demonstrated the decrease in protein-carbonyl levels of several identified proteins leading to an increase in glycolytic enzyme activities and fermentation properties. References: 1. Pe´rez-Torrado et al. (2009) Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth. Appl Microbiol Biot 81:951–960. 2. Go´mez-Pastor et al. (2010) Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass. Microb Cell Fact 9:9. 3. Go´mez-Pastor et al. (2010) Transcriptomic and proteomic insights of the wine yeast biomass propagation process. FEMS Yeast Research 10: 870–884.


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Abstracts YSF.41 Atomic level description of the domain closure in a dimeric enzyme: thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) E. Graćzer1, A. Merli2, R. K. Singh3, M. Karuppasamy4, P. Za´vodszky1, M. S. Weiss5 and M. Vas1 1 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary, 2Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy, 3National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, India, 4Leiden University Medical Center, Molecular Cell Biology, Electron Microscopy Section, Leiden, the Netherlands, 5 Helmholtz-Zentrum Berlin fu¨r Materialien und Energie, Macromolecular Crystallography (BESSY-MX), Berlin, Germany Introduction: A so-called ‘‘hinge bending’’ motion, i.e. opening and closing of two respective domains, has been observed in a number of proteins, both monomeric and oligomeric. Isopropylmalate dehydrogenase molecule’’ consists of two identical subunits and each of the subunits can be divided into two structural domains. Comparison of various known crystal structures of IPMDHs indicates that the relative position of the two domains may vary between open and closed. This suggests that domain closure plays an essential role during the catalytic cycle, similar to the mechanisms proposed also for other multidomain enzymes. Methods: Different substrate-IPMDH complexes were determined by X-ray crystallography. Molecular graphical analysis was used to describe a possible molecular mechanism of substrate-induced conformational transitions as well as the structural background of the catalysis by IPMDH. Results and Conclusions: Mapping of all contacts of the conserved side chains and their changes upon substrate binding have led us to identify two notable hinges: hinge 1 in the turn connecting a-helix d and the b-strand F as well as hinge 2 in the loop between the a-helix h and the following b-strand E. The analysis has also revealed that the substrate Mn2+/IPM complex has an essential role in operation of these hinges. NADH also influences the domain closure, but in a much smaller extent. In addition, the subunit-subunit contacts formed in the cavity between the two domains were also found to contribute to the domain motions. Thus, the structure-function relationship of IPMDH provides an example of high extent of cooperativity between domains and subunits.

YSF.42 Phosphorylation of human centrins 1 and 2 by PKA and CK2 protein kinases D. Grecu, Y. Blouquit and Liliane Assairi Institut Curie, Inserm U759 Centrins are member of the EF-hand superfamily of calciumbinding proteins. Centrins bind to several cellular targets : XPC (involved in NER), Sfi1 (involved in the centrosome duplication), transducin (involved in the phototransduction cascade) via a helicoidal and hydrophobic motif W1L4L8 in the case of XPC. The dipole of this motif has a reverse orientation such as L8L4W1 in the case of Sfi1 and transducin. This inversion has for consequence a slight translation of the motif within the hydrophobic cavity of centrin (1). Centrins are phosphorylated by several protein kinases. PKA phosphorylates human centrins 1 and 2 at the Ser170 (2). A sec-

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YSF – Young Scientist Forum ondary phosphorylation site has been identified. PKA phosphorylation of centrins is inhibited in the presence of calcium. The analysis by circular dichroism provides almost the same profile of the secondary structure for both non-phosphorylated and phosphorylated centrins, in presence and in absence of calcium. The binding capacity to targets (XPC, Sfi1) of PKA-phosphorylated centrins 1 and 2 as well as of their variant S170D are analyzed by Isothermal Titration Microcalorimetry (ITC). CK2 phosphorylates human centrins 1 and 2 at the Thr138, and reduces the binding of centrin to transducin (3). The binding capacity to transducin of CK2-phosphorylated centrins1 and 2 and of their variant T138D is explored by ITC. References: 1. Martinez-Sanz, Kateb, Assairi, Blouquit, Bodenhausen, Abergel, Mouawad, Craescu (2010) J. Mol. Biol. 395, 191–204. 2. Lutz, Lingle, McCormick, Greenwood, Salisbury (2001) J. Biol. Chem. 276, 20774–20780. 3. Thissen, Krieglstein, Wolfrum, Klumpp (2009) Research Letters in Biochemistry.

YSF.43 Characterisation of the PARP12 novel ADP- ribosyltransferase activity and of its cellular substrates G. Grimaldi, G. Catara, C. Valente, A. Flagiello, P. Pucci and D. Corda CNR Institute of Protein Biochemistry, Naples, ItalyCEINGEBiotecnologie Avanzate, Via Comunale Margherita, Naples ADP-ribosylation is an ubiquitous protein modification that controls many cellular processes, including transcription, DNA repair and bacterial toxicity. This reaction involves the transfer of a single ADP-ribose moiety (mono-ADP-ribosylation) or elongated and branched ADP-ribose polymers (poly-ADP-ribosylation) to a specific amino acid of the acceptor protein and can be catalysed by ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs), respectively. The PARP family can be divided into three classes: one representing bona fide PARP enzymes with poly-ADP-ribose polymerase activity like PARP1, a second one representing PARP-related mono-ADP-ribosyltransferases (mARTs), and a third one without enzymatic activity due to the lack of critical residues in the NAD+ binding fold. To gain insight into the different biological functions displayed by mono versus poly-ADP-ribosylating enzymes in cells, we are focusing our attention on the potential PARP-related mARTs. Here we report our recent data related to PARP12, indicating that this is a novel mono-ADP-ribosyltransferase, that can modify acidic residues. To elucidate its biological role, we analysed its intracellular localisation and identified its substrates. Immunofluorescence experiments have indicated that PARP12 is localised on the Golgi complex and it represents the first mARTs localised on this organelle. Moreover, using an affinity purification method based on a protein module (the macro-domain) that recognizes ADP-ribosylated proteins, together with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we identified 25 putative substrates, and clustered them according to their cellular function. By validating these substrates, our preliminary data indicate an involvement of PARP12 in RNA processing and membrane trafficking.


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Abstracts

YSF.44 Different membrane interactions of C2 domains

YSF.46 Crystal structures of human sulfotransferase 1A1: from broad to narrow specificity

J. Guilleń, G. Luengo-Gil, M. Guerrero-Valero, T. CoronadoParra, J. C. Go´mez-Fernańdez and S. Corbalań-Garcı´ a Bioquı´mica y Biologıá Molecular A, Universidad de Murcia, Murcia, Spain

C. Guttman, I. Berger, A. Aharoni and R. Zarivach 1 Departments of Life Sciences, Ben-Gurion University of the Negev, Be’er Sheva, Israel, 2National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be’er Sheva, Israel

C2 domains are a ubiquitous conserved Ca2+-activated membrane-docking modules present in a wide range of Ca2+-regulated proteins. Several evidences have demonstrated that some of these domains are able to interact with PtdSer and with the inositol phospholipid PtdIns(4,5)P2, which is able to directly participate in a myriad of functions, including cell signaling at the plasma membrane, regulation of membrane traffic and transport, cytoskeleton dynamics, and nuclear events. Currently, much evidence has supported the idea that C2 domains can interact with lipids using two separate binding sites, the Ca2+-binding loops and the cationic b-groove. When we analyzed the similarity of different C2 domains by structure-based sequence alignment a potential PtdIns(4,5)P2 interacting-site can be predicted. Despite the numerous studies, a significant degree of disagreement exists in the literature about the lipid selectivity of C2 domains. In the present work we analyzed the different lipid specificity of several C2 domains. The binding to Ca2+ and different membrane model systems composed of PtdIns(4,5)P2 and/or PtdSer have been characterized by using isothermal titration calorimetry and fluorescence spectroscopy. This work was supported by grants from Fundacioń Seńeca 08700/PI/08 (to S.C.-G.), and Ministerio de Ciencia e Innovacioń BFU2008-01010 (to J.C.G.-F.). J.G.-C. belongs to the Programa Juan de la Cierva from the Ministerio de Ciencia e Innovacion.

YSF.45 Studying the role of SAGA deubiquitination module in transcription regulation Y. D. Gurskiy, D. V. Kopytova and S. G. Georgieva Institute of Gene Biology, Russian Academy of Sciences (RAS), Moscow, Russia SAGA is a multiprotein transcriptional coactivator complex that is conserved across eukaryotes. It reveals multiple functions during transcriptional activation and plays important roles in the regulation of expression of a subset genes, transcribed by Pol II. SAGA is involved in chromatin remodeling through its catalytic subunit GCN5 (acetylation of histones) and the other separate subcomplex called the deubiquitination module (DUBm). Drosophila DUBm consists of the Nonstop, dSgf11, dENY2, and functions as deubiquitinating machinery of histone H2B, thereby it provides a pattern of modifications that are essential for efficient regulation of gene expression. We rised specific antibody against Drosophila DUBm components. Using antibody against Nonstop, dSgf11 and dENY2 we studied DubM localization in cell and on different sites of polythene chromosomes. We also demonstrated a distribution of components of DUBm in distinct fractions of nuclear extract from Drosophila embryos and its association with various regulatory elements of Drosophila genome.

Human Sulfotranferase 1A1 (hSULT1A1) catalyzes the transfer of a sulforyl group from a 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) donor to a variety of substrates (acceptors) containing either an amine or a hydroxyl group, leading to modification of the acceptor’s biological activity. In order to gain insight into the molecular mechanism underlying the broad specificity of hSULT1A1, we have crystallized the enzyme and determined its structure in the presence of 3¢ phosphoadenosine 5¢-phosphate (PAP) alone, PAP and 3-Cyano-7-Coumarin (3CyC), and PAP and 2-Napthol (2NAP). These structures demonstrated high plasticity of the acceptor binding site which was mainly attributed to substantial movements of the gating loop (residues 86–90) that enabled the binding of large and elongated phenol substrates. We were also interested in increasing the SULT1A1 specificity to para-nitrophenol (pNP) on the account of the other substrates through the use of directed evolution methodology. This method allowed the generation of a hSULT1A1 variant mutated at position D249G which exhibits a marked increase in activity toward pNP while decreasing its activity toward 3CyC and 2NAP. The determined crystal structure of D249G revealed the effect of the mutation on surface electrostatic potential and loop stability in the proximity of the active site pocket.

YSF.47 Link between the key lipogenic enzyme ATP citrate lyase and hepatic receptor for triglyceride-rich lipoproteins, the lipolysis stimulated lipoprotein receptor M. Hanse, C. Stenger, S. Akbar, C. Malaplate-Armand, J.-L. Olivier, T. Oster and F. T. Yen Lipidomix laboratory, Nancy University INPL ENSAIA Hepatic lipolysis stimulated lipoprotein receptor (LSR) plays an important role in the removal of triglyceride (TG)-rich lipoproteins during the postprandial phase. Recent data in our laboratory revealed that the adipokine leptin regulates LSR expression, accompanied by significant changes in hepatic lipid metabolism (Stenger et al, 2010). In this study, we sought to determine if a direct link exists between LSR and de novo lipid synthesis in the liver. A key lipogenic enzyme, ATP citrate lyase (ACL), converts citrate to acetyl-CoA, the substrate for de novo fatty acid synthesis. Hydroxycitric acid is a potent inhibitor of ACL found in Garcinia cambogia. Female C57Bl/6Rj mice were treated daily for 40 days by gavage with a corn oil/physiological saline emulsion alone or containing 20 mg of Garcinia cambogia extract (GCE). As reported in the literature, inhibition of ACL led to a significant decrease (50%, p = 0.03) in plasma TG and decreased body mass in GCE-treated mice with no significant change in food intake. Immunoblots revealed that total liver membrane LSR protein levels were significantly increased by two-fold in the GCE-treated mice. Hepatic TG content was increased by 20% in GCE-treated mice, which may be due to increased influx of exogenously-derived lipids through the LSR pathway. Interestingly, both liver lipoprotein and hepatic lipases were significantly decreased (27%, p = 0.05; 38%, p = 0.04, respectively), indicating a potential feedback regulation to limit overloading the liver


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Abstracts with an excess lipid load. Indeed, LSR activity as a lipoprotein receptor is dependent on the production of lipolytic products, free fatty acids. We propose that when de novo fatty acid synthesis is inhibited, LSR expression is increased in order to provide the liver with the necessary energy substrate in order to compensate for the lack of endogenous supply of lipids, suggesting that LSR is closely linked with the metabolic events involved in maintaining energy homeostasis.

YSF.48 New players in recognition of intact and cleaved apurinic/apyrimidinic sites: searching and identification with AP DNA E. S. Ilina, M. V. Sukhanova, M. M. Kutuzov, O. I. Lavrik and S. N. Khodyreva Institute of Chemical Biology and Fundamental Medicine SB RAS One of the most abundant DNA lesions are apurinic/apyrimidinic sites (AP sites) arising as a result of hydrolytic cleavage of Nglycosylic bonds spontaneously or through glycosylase-catalyzed removal of damaged bases during the early stage of base excision repair. The number of AP sites can increase dramatically under stressful conditions such as X-ray or UV light irradiation or oxidative and alkylating agent exposure. Some proteins participating in processing of AP sites form Schiff base with deoxyribose of AP site, which can be easily converted to a stable product by sodium borohydride treatment (Schiff base mediated cross-linking). This approach was used to search in mammalian cell extracts proteins that specifically interact with AP sites. By combination of cross-linking technique with MALDI-TOF-MS or immunochemical methods, poly(ADP ribose) polymerase 1 (PARP1) and Ku80 subunit of Ku antigen were for the first time identified as proteins reactive to AP site. Both PARP1 and Ku80 are responsible for cellular radioresistance. The level of Ku80 cross-linking to AP DNA was shown to be a relevant factor for estimation of the Ku80 content in human cell extracts. PARP1 interaction with AP sites (intact or cleaved) via the same mechanism was shown to be involved in the lyase activity of the enzyme. AP-DNA duplexes with additional lesions located opposite AP sites were used to mimic clustered damages. By interaction with AP sites PARP1 and Ku antigen modulate the AP site hydrolysis by apurinic/apyrimidinic endonuclease 1, with PARP1 being more effective as the inhibitor at DNA duplexes with additional lesions. Chemically reactive DNAs appear to be a versatile tool in identification of proteins interacting with particular DNA intermediate. This work was partially supported by program of RAS ‘‘Molecular and Cellular Biology’’, RFBR projects 09-04-93106, 10-0401083.

YSF.49 HDAC inhibitors reduce the expression of markers involved in endothelial lineage progression F. Iordache, C. Buzila, E. Andrei, A. Constantinescu, M. Simionescu and H. Maniu Institute of Cellular Biology and Pathology ‘‘Nicolae Simionescu’’, Bucharest, Romania To test the involvement of histone deacetylases (HDACs) activity in endothelial lineage progression we investigated the effects of HDAC inhibitors on endothelial progenitors cells (EPCs) derived

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YSF – Young Scientist Forum from umbilical cord blood (UCB). Adherent EPCs, that expressed the endothelial marker proteins (PCAM, CD105, CD133, and VEGFR2) revealed by flow cytometry were treated with three HDAC inhibitors: Butyrate (BuA), Trichostatin A (TSA), and Valproic acid (VA). RT-PCR assay showed that HDAC inhibitors down-regulated the expression of endothelial genes such as VE-cadherin, CD133, CXCR4 and Tie-2. Furthermore flow cytometry analysis revealed that HDAC inhibitors selectively reduce the expression of VEGFR2, CD117, VE-cadherin, and ICAM-1, whereas the expression of CD45 and CD34 remained unchanged, demonstrating that HDAC is involved in endothelial differentiation of progenitor cells. Real-Time PCR showed that TSA down-regulated telomerase activity probably via suppression of hTERT expression, suggesting that HDAC inhibitor decreased cell proliferation. Cell motility was also decreased after treatment with HDAC inhibitors as shown by wound-healing assay. In conclusion, the balance of acethylation/ deacethylation kept in control by the activity of HAT (histone acetyltransferases)/HDAC enzymes play an important role in differentiation of stem cells by regulating proliferation and endothelial lineage commitment.

YSF.50 Structural studies of amino acid:[carrier protein] ligase in the complex with carrier protein N. Ivic1, M. Mocibob2, I. Weygand-Durasevic2 and M. Luic1 1 Division of Physical Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia, 2Department of Chemistry, University of Zagreb, Faculty of Science, Zagreb, Croatia Aminoacyl-tRNA synthetases (aaRS) are enzymes responsible for attachment of amino acids to the cognate tRNA molecules, which makes them essential for ribosomal protein biosynthesis. Recent investigations discovered numerous aaRS-like proteins in a wide range of organisms although aaRSs are evolutionary highly conserved. We have recently identified and characterised bacterial homologues of atypical arcaeal seryl-tRNA synthetases (aSerRS). These aSerRS homologues do not aminoacylate tRNA. Instead, they covalently attach amino acid to phosphopantetheine prosthetic arm of the cognate carrier protein (CP) and function as amino acid:[carrier protein] ligases. In comparison to SerRSs, they show different amino acid specificity, activating Gly (or Ala) instead of Ser. The crystal structure of glycine: [carrier protein] ligase (Gly:CP ligase) Bll0957 from Bradyrhizobium japonicum shows remarkable similarity to catalytic domain of aSerRS as well as the active site topology. Serine-ordering loop responsible for Ser binding into the active site of aSerRS is replaced by a loop-helix motif located further away from the active site. To understand the binding mode of CP to aSerRS homologue, the crystal structure of Gly:CP ligase in a complex with its cognate carrier protein was recently solved. The carrier protein binds to the helix of the idiosyncratic loop-helix motif of Gly:CP ligase mostly through hydrophobic interactions. Only two Gly:CP ligase residues (Arg220 and Glu231) form hydrogen bonds with the CP. The phosphopantetheine group of carrier protein enters into the active site of the Gly:CP ligase through a wide tunnel from the opposite side than tRNA to aSerRS active site. The structure of Gly:CP ligase in complex with its cognate carrier protein provides insight into how these aSerRS homologues recognize fundamentally different macromolecular substrate.


YSF – Young Scientist Forum YSF.51 Bacteriophage SPP1 infection of Bacillus subtilis: evidence for a preferential polar route for entry in a Gram-positive bacterium L. Jakutyte1,2,3, C. Baptista4,5, C. Saõ-Jose´4,5, R. Daugelavicˇius3, R. Carballido-Lopez2 and P. Tavares1 1 Unite´ de Virologie Molećulaire et Structurale, CNRS UPR3296 and IFR 115, Baˆtiment 14B, CNRS, Gif-sur-Yvette, France, 2 Institut National de la Recherche Agronomique, Unite´ Mixte de Recherche 1319 Micalis, Domaine de Vilvert, Jouy-en-Josas, France, 3Department of Biochemistry and Biophysics, Vilnius University, Cˇiurlionio 21, Vilnius, Lithuania, 4Instituto de Medicina Molecular, Av. Prof. Egas Moniz, Ed. Egas Moniz, Lisboa, Portugal,5Unidade dos Retrovirus e Infecço˜es Associadas, Centro de Patogeńese Molecular, Faculdade de Farmaćia da Universidade de Lisboa, Avenida das Forças Armadas, Lisboa, Portugal Entry in bacteria is one of the less understood steps of bacteriophages life cycle. The different envelopes of Gram-negative bacteria, with its fluid outer membrane, and Gram-positive bacteria, exposing a thick peptidoglycan wall to the environment, impose distinct challenges for bacteriophage binding and dynamics at the host cell surface. Infection of the Gram-positive bacterium Bacillus subtilis by bacteriophage SPP1 was followed in space and time. SPP1 binds preferentially to cell poles. This topology favors irreversibly adsorption to the SPP1 phage receptor protein YueB, a component of a putative type VII secretion system. YueB is found to concentrate at the bacterium poles and displays a helical-like distribution at the B. subtilis surface. The dynamics of SPP1 DNA entry and replication were visualized in real time. During infection, most of the infecting phages DNA entered and replicated at a position of the host cytoplasm near the cell poles. This cell biology study on Gram-positive bacteria infection indicates that the topology of SPP1 receptors determines the position of DNA entry in the cell and subsequently the site of its replication which occurs in discrete foci.

YSF.52 Biochemical characterisation and proteomic profiling of Cronobacter strains isolated from different food samples B. Javu˚rkova´, M. Blazˇkova´, P. Junkova´, L. Fukal and P. Rauch Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, The Institute of Chemical Technology, Prague, Czech Republic Cronobacter spp. (formerly Enterobacter sakazakii) are opportunistic foodborne pathogens from the family Enterobacteriaceae. These strains are ubiquitous and cause rare but severe infection among neonates and infants. Outbreaks of Cronobacter infections are mostly associated with consummation of contaminated powdered infant formula. In the present study, infant formula and food samples were screened for the presence of Cronobacter spp. and different biochemical principles were used for their characterisation. Genus and species classification were provided by PCR targeted to 16S rDNA and rpoB gene, respectively. The isolated strains were further biochemically characterized via the bioMe´rieux ID 32E and additional biochemical tests. In order to study the similarity of the isolates the SDS–PAGE electrophoresis fingerprints of cell extracts were compared. The characteristic bands of proteins were identified by peptide-mass mapping analysis.

Abstracts Acknowledgement: this work was supported by the Czech Grant Agency (project no. 525/09/1075), Ministry of Education of Czech Republic (projects nos. MSM6046137305 and 2B06048), and Specific University Research (MSMT No.21/2011).

YSF.53 Casein kinase-1e and circadian gene expression and its ischemic and hypoxic regulation in glioma cells are dependent from inositol requiring enzyme-1a gene function L. L. Karbovskyi, D. O. Minchenko, M. Moenner and O. H. Minchenko Department of Molecular Biology, Palladin Institute of Biochemistry National Academy of Sciences of Ukraine, Kyiv, Ukraine, Department of Pediatrics, Bohomoletz National Medical University Academy of Medical Sciences of Ukraine, Kyiv, Ukraine,INSERM U920 Molecular Mechanisms of Angiogenesis Laboratory, University Bordeaux 1, Talence, France. Astrocytes represent the most abundant cell type in mammalian brain, and play an important role in the maintenance and regeneration of neuronal functions. Hypoxia and glucose deprivation, which are essential features of ischemia and chemicals have been shown to induce a set of complex intracellular signaling events known as the unfolded protein response. This adaptive response is mediated by three endoplasmic reticulum-resident sensors but inositol requiring enzyme-1a is a central mediator of the unfolded protein response. The blockade of inositol-requiring enzyme-1a serine/threonine kinase and endoribonuclease activities is thought to either lead to a decrease in survival signals between astrocytes and neuronal cell, as suggested by transcriptomic analyses. The main goal of this work is to study the effect of ischemic conditions on the expression of casein kinase-1e, Cry1, Clock, BMal1b and BMal2 genes in glioma cell line U87 and its subline with inositol-requiring enzyme-1a deficiency. We provide evidence that inhibition of inositol-requiring enzyme-1a signaling results in the reduction in the tumor proliferation index and prolongs overall survival of glioma-implanted mice. We have shown that the expression of casein kinase-1e, casein kinase-d, Cry1 and BMal2 mRNA is significantly decreased but Clock mRNA is decreased in U87 cells with inhibition of enzymatic activities of inositol-requiring enzyme-1a. Hypoxia decreases the expression of Clock, Cry1 and BMal2 mRNA but increases BMal1b and casein kinase-1e. Ischemic condition did not change significantly expression of Clock and Cry1 but induces the expression of BMal1b, BMal2 and casein kinase-1e and d. Thus, our results showed that reduction in the proliferation rate of glioma cells with inhibition of inositol-requiring enzyme-1a function possibly connected with deregulations of circadian clock system.

YSF.54 Identification, cloning and expression of a human RNase j isoform arising from subtle alternative splicing E. D. Karousis, E. G. Fragoulis and D. C. Sideris Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Athens, Greece Human RNase j is a novel endoribonuclease, expressed in normal and malignant tissues in all developmental stages [1]. It belongs to a recently identified, highly conserved in metazoans protein family whose biological role is currently under investigation.


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Abstracts Northern blot experiments revealed the expression of a main transcript (approx. 700 bp) in all human cell lines examined, while an EST database analysis led to the retrieval of an additional sequence subset lacking four consecutive bases compared to the original RNase j mRNA. In order to isolate this putative isoform, we excluded the original RNase j transcript from the cDNA pool by combining an RT-PCR hybrid selection strategy followed by complete digestion using the appropriate restriction enzyme. By this approach we cloned an alternative transcript (RNase j-02) harboring an ORF of 405 nucleotides, encoding a 134 amino-acids protein. Compared to the original RNase j (98 a.a.), the 63–134 a.a. region of RNase j-02 is absolutely identical to 27–98 a.a. of RNase j, whereas the 1–62 aminoterminal portion bears no similarity. The difference in primary structure results from the usage of an upstream start codon in combination with a subtle alternative splicing event occurring in the exons 1–2 junction. RNase j02 mRNA is expressed in a series of human cell lines (HEK-293, HeLa, SKOV-3, Jurkatt, BL41, FM3, U251, SY5Y, BT-20, HT29) as verified by applying a modified RT-PCR approach. In order to characterize the RNase j-02 protein, the yeast P. pastoris expression system has been employed, the expression of the recombinant protein has been verified and recent findings indicate that RNase j-02 isoform exhibits endoribonucleolytic activity. Reference: 1. Economopoulou M.I. et al. (2007) Nucleic Acids Research. 35, 6389–6398.

YSF.55 Structure-functional analysis of yeast Rpn4, a transcriptional regulator of 26S proteasome D. Karpov, T. Vera and K. Vadim Engelhardt Institute of Molecular Biology RAS The 26S proteasome is a multi-subunit protease complex which plays an essential role in many basic cellular processes. The abundance of the 26S proteasome is controlled by a negative feedback circuit that involves the Rpn4 – an extremely shortlived transcriptional factor and proteasome substrate. While mechanisms of proteasome-dependent Rpn4 degradation are well studied, much less is known about mechanisms of Rpn4-dependent proteasome regulation. We performed a systematic deletion analysis of Rpn4 to map its functionally important domains. Rpn4 deletion derivatives were expressed in rpn4- yeast strain and characterized in several ways. First, expression levels of mutant proteins were determined by Western blotting. Second, overall activity of proteins was estimated by measuring mRNA level of proteasomal genes. Third, the DNA-binding activity of Rpn4 derivatives was assessed by using chromatin immunoprecipitation assay. Our results show that deletion of C-terminal region containing DNA-binding domain inactivates Rpn4. Putative nuclear localization signals are not required for Rpn4 function. However, N-terminal region containing degrons and sites of polyubiquitination is crucial for Rpn4 transactivation function. Within the N-terminus we have found a novel transactivation domain that shares no common features with any known class of transactivation domains. Mutation of all four lysines to arginines within this domain creates a hyperactive form of Rpn4 and does not affect its expression level. Hyperactivated Rpn4 is toxic to cells under stress but not physiological conditions. These results suggest that C-terminal and N-terminal domains are important for Rpn4 activity. Moreover, Rpn4 transactivation potential might be negatively regulated through lysines in the N-terminal domain under stress conditions in a proteolysis-independent manner. This work was supported by the Russian foundation for basic research and the Grant from the President of Russian Federation.

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YSF – Young Scientist Forum YSF.56 (S12.2.5) Does activation of DNA damage response in human T cells leads to cellular senescence? Z. Korwek, A. Bielak-_zmijewska, G. Mosieniak, O. Alster and E. Sikora Laboratory of Molecular Bases of Ageing, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland Cellular senescence, described for the first time almost five decades ago as replicative senescence, is characterized by loss of the proliferative potential, which is the outcome of telomere shortening after each cell division. Now it is known that cellular senescence can be induced by different stress factors in the process called stress induced premature senescence (SIPS). An important role in both types of senescence plays DNA damage, which activates the DNA damage response (DDR) signaling pathway. The key protein is p53, which can lead to cell cycle arrest and senescence by the induction of p21 or to apoptosis by the induction of Bax .The aim of our study was to investigate the molecular mechanism of cellular senescence and apoptosis of normal human T cells and the role of DNA damage in these processes. First, we checked the response of quiescent T cells to DNA damage induced by etoposide. We observed the expression of the marker of DNA double strand breaks cH2AX, and upregulation of phosphorylated proteins involved in DDR, such as: ATM, Chk2 and p53. Also proapoptotic Bax activation was observed. Altogether, in quiescent T cells DNA damage induced by etoposide activated DDR leading to apoptosis. To answer the question whether DNA damage can be involved in T cell senescence we stimulated cells with a unspecific mitogen PHA, which mimics TCR activation. PHA stimulation caused hyperproliferation, which induced replication stress and the DNA damage as observed by cH2AX foci formation. Moreover, we found upregulation of p53 and p21, and senescence associated b-galactosidase activity, a marker of senescent cells. Summing up, replicative stress just like direct DNA damage leads to DDR, but only hyperproliferation causes both cell death and cellular senescence.

YSF.57 Extra residue in double stranded hinge region of ribosomal protein L1 from Thermus thermophilus is responsible for its closed conformation O. Kostareva, S. Tishchenko, E. Nikonova, A. Gabdoulkhakov, N. Nevskaya, M. Garber and S. Nikonov Institute of Protein Research, Russian Academy of Sciences Ribosomal protein L1 is an important primary 23S rRNA-binding protein as well as a translational repressor binding its own mRNA. Crystal structures of bacterial L1 from Thermus thermophilus (ThL1) and archaeal L1 from Methanococcus jannaschii (MjaL1) and their complexes with specific fragments of mRNA were determined earlier in our laboratory. L1 protein consists of two domains connected by a flexible double-stranded hinge. The overall shapes of archaeal and bacterial L1 proteins in isolated form differ dramatically because of the different relative positions of the domains. Archaeal L1 proteins have the so-called open conformation, closed conformation is characteristic of bacterial proteins. TthL1 complexed with the fragment of mRNA has an open conformation like that of archaeal L1. Analysis of all known L1 sequences shows that the hinge region of bacterial L1 proteins differs from corresponding regions of L1 from other sources by an extra residue in one of two oppositely directed polypeptide chains. To test whether an additional residue


YSF – Young Scientist Forum in the hinge could change the conformation of a two-domain molecule, we obtained mutant TthL1 lacking Ala in the hinge region and mutants MjaL1 with extra Ala or Lys in a corresponding position. These mutant forms were crystallized, and their crystal structures were determined. It was shown that the lack of Ala switches the closed conformation of L1 to an open one. Therefore we may suggest that just this Ala is responsible for the closed conformation of TthL1 and that all bacterial L1 must have closed conformations in an isolated form due to an extra residue in the hinge region. Mutants MjaL1, containing an extra residue in the hinge region, hold an open conformation although the domains are brought closer together. This research was supported by the Russian Academy of Sciences, the Russian Foundation for Basic Research (No. 10-0400618), the Program of RAS on Molecular and Cellular Biology, the fellowships from FEBS and EMBO.

YSF.58 Changes of activity and glycosylation of fibronectin in myocardial infarction A. Kulinich, G. Maslak, I. Pismenetskaya and A. Shevtsova Dnipropetrovsk State Medical Academy Fibronectin (FN) is a multifunctional glycoprotein of blood plasma and extracellular matrix that have several sites of glycosylation. Carbohydrate component can influence protease-resistance and functional activity of FN. Alterations of FN glycosylation were reported in some pathological states but not in myocardial infarction. Aim: To investigate FN glycosylation and its functional activity in myocardial infarction. Matherials and methods: Level of FN and its functional activity were investigated in blood plasma of 26 patients with myocardial infarction and 20 healthy donors (control) with immuno-dot analysis and cold heparin-precipitation. Glycosylation of FN was studied with lectin-enzyme analysis using deglycosylated antibody to FN, fucose – specific (Lens culinaris agglutitnin – LCA, Aleuria aurantia lectin – AAL) and sialic acid- specific (Sambucus nigra agglutinin –SNA, Maacia amurensis agglutinin – MAA) lectins. Results: The level of FN did not change substantially in myocardial infarction in comparison with the control. Heparin-binding activity of FN increased 2-fold reflecting changes of functional activity of FN. Decreasing of FN affinnity to some lectins was detected. Binding of FN with the lectins was at the average 30% lower in patients with myocardial infarction by contrast to the control. The lowest values (34.4 ± 7.1%) were registered in interaction with AAL. Negative correlation(r = 0.89, p < 0.5) between biological and AAL-binding activity of FN was revealed. Conclusion: Glycosylation and functional activity of FN change in myocardial infarction. Taking into account the role of FN in cell-cell interactions and in survival of myocardiocytes these alterations can be crucial in the disease development. The results obtained can be useful in diagnostics of cardiovascular diseases.

Abstracts YSF.59 Mechanism of DNA repair by 8-oxoguanineDNA-glycosylase N. Kuznetsov and O. Fedorova Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia Reactive oxygen species damage DNA to produce a variety of genotoxic lesions. In particular, 7,8-dihydro-8-oxoguanine (oxoG) is one of the most common pre-mutagenic products of base oxidation in DNA. OxoG is repaired through excision by 8-oxoguanine-DNA glycosylases. It is still unclear how DNA glycosylases robustly select sparse lesions among the vast excess of normal DNA. One possibility is that the conformational transitions during substrate binding occur sequentially, with at least some steps structurally optimized for selection of correct substrates. This model was supported by stopped-flow studies of Fpg from E. coli. The five-step kinetic scheme was revealed by the protein internal Trp fluorescence for binding with oxoG-substrate indicating multiple conformational transitions in enzyme. The parallel analysis of 2-aPu fluorescence showed that these steps correspond to only two fluorescently discernible conformational changes in DNA. The direct measurement of pre-steady-state binding and bending of DNA substrates labeled with Cy3 and Cy5 FRET-dyes as well as ESI/MS intermediate detection during time course of reaction lead in the conclusion that the regeneration of the free enzyme from its conjugate with ribose residue of DNA substrate most likely occurs after the dissociation of the enzyme–product complex and limits the reaction rate under multiple-turnover conditions. Support from RFBR (10-04-00070), SB RAS (48) and Russian Ministry of Education and Science (3185.2010.4, 1304.2010.4) is acknowledged.

YSF.60 The effect of metal ions coupled with synaptic signalling on sod1 aggregation events S. S. Leal1,2, J. S. Valentine2 and C. M. Gomes1* 1 Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal, 2Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA Cu/Zn superoxide dismutase (SOD1) is a dimeric protein implicated in amyotrophic lateral sclerosis (ALS). Aggregation events are a hallmark of this fatal neurodegenerative and although ubiquitously expressed in all tissues, SOD1 only aggregates specifically in motor neuron cells of ALS patients. This evidence suggests that circumstantial conditions to neural tissues activity are likely mandatory for the onset of SOD1 aggregation pathology. In this respect, high levels of free zinc and calcium ions are a very specific feature of neuronal synaptic traffic, suggested to be deregulated in ALS patients and to play a role in neurodegeneration. Presently, we are investigating the effect of zinc bursts and calcium ions on the SOD1 structural features and related aggregation events, using biophysical methods. Here we will report our results suggesting that at increasing concentrations of these metal ions, apoSOD1 undergoes particular conformational alterations and that this is followed by a significant increase in the propensity to form non-native oligomeric species. This effect is accompanyed by a significant change in the relative content in secondary structure of SOD1, noticeably an increment of the intermolecular b -sheet aggregation band. These observations illustrate how even a transient imbalance in cellular zinc and calcium concentrations may trigger the formation of toxic SOD1 aggregates and contribute for ALS neurotoxicity.


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Abstracts YSF.61 In CD28-costimulated human nave CD4+ T cells IKK/NF-jB signaling pathway promotes cell proliferation through IL-2-independent mechanisms E. Lupino, B. Buccinna`, C. Ramondetti, A. Lomartire, G. De Marco, M. T. Rinaudo and M. Piccinini Department of Medicine and Experimental Oncology, section of Biochemistry, University of Turin, Turin, Italy Stimulation of nave CD4+ T cells through engagement of the Tcell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signaling via the IL-2 receptor (IL2RA). However, several studies indicate that in CD28 co-stimulated T cells additional IL-2-independent signals are also required for cell proliferation. Aim of this study was to determine if IKK/NF-jB signaling pathway promotes T cell proliferation independently of the pivotal role it retains in the up-regulation of IL-2 and IL-2RA gene expression. The effects of two selective, structurally unrelated, cell-permeable IKK inhibitors, BMS-345541 and PS-1145, were evaluated in parallel with those elicited by a neutralizing anti-human IL-2 antibody on the expression of cell-cycle regulatory proteins in TCR/CD28 co-stimulated human nave CD4+ T cells. We demonstrate that in these cells IKK/NF-jB signaling pathway controls the expression of cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms. These findings underscores a crucial function of IKK/NF-jB signaling pathway in the regulation of T cell entry into S phase. In conclusion these results provide new information about the molecular basis of the immunosuppressive and anti-inflammatory effects of IKK inhibition, although further work is needed to thoroughly understand the mechanisms by which IKK/NF-jB signaling pathway regulates the proliferation of human nave CD4+ T cells.

YSF.62 Comparative study of native and truncated CP4 5-enolpyruvylshikimate-3-phosphate synthase gene to assess horizontal gene transfer from genetically modified food M. Madyagol, P. Utekal, S. Stuchlı´ k and J. Turnˇa Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska´ dolina, Bratislava, Slovak Republic Public and scientific concerns about the environmental and food safety of genetically modified (GM) crops overshadow the potential benefits offered by crop biotechnology to improve food quality. One of the main concerns regarding the use of GM food in human and animal nutrition is the effect that newly introduced sequences may have on the organism. The main objective of our study is to quantify possible horizontal gene transfer (HGT) from

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YSF – Young Scientist Forum the GM plants to bacterial microflora. For studying potential gene transfer we have chosen CP4 EPSPS (5-enolpyruvylshikimate 3-phosphate synthase) gene from Round Ready maize chromosome. The CP4 EPSPS enzyme is a key enzyme in the biosynthesis of aromatic amino acids in microorganisms and plants. It is the target of the broad-spectrum herbicide glyphosate (marketed under the trade name Roundup). During our earlier studies of HGT of the CP4 EPSPS gene from GM maize through gastrointestinal tract to bacteria living in animal gut we have observed a functional truncated CP4 EPSPS gene form present in bacteria. The native and truncated CP4 EPSPS genes were cloned into the T7 expression vector pET-28b(+). We have tested the truncated form of CP4 EPSPS indetail and carried a comparative study with native full-length CP4 EPSPS. We have observed highest level of protein expression of both forms of CP4 EPSPS synthases. The expressed protein of size 47 kDa for full length and 42 kDa for truncated CP4 EPSPS respectively was purified. In our future work we will characterize and study enzyme activity of truncated CP4 EPSPS and compare with native enzyme. This will help us to decide that some truncated forms of CP4 EPSPS could confer full function of native full-length CP4 EPSPS and these findings also should be taken into account in risk assessment of possible HGT from GM plants.

YSF.63 P{EP}EP3171 element remobilization in Drosophila melanogaster induced both DmManf and CG14879 mutant alleles L. M. Magdalena1, L. Savu2 and I. Kiss3 1 Department of Genetics, University of Bucharest, Bucharest, Romania, 2Genetic Lab SRL, Bucharest, Romania, 3Institute of Genetics, Biological Research Centre, Szeged, Hungary Transposition of the mobile elements derivatives plays an important role in the generation of genotypic and phenotypic diversity in D. melanogaster. Using the 2-3 transposase source we carried out a local mutagenesis in the 89B13 chromosomal region where the P{EP}EP3171 element was located. Characterization of the P{EP} excision products in molecular and phenotypic terms revealed that the repair process of the DNA double strand break induced by the remobilization yielded to different types of excision products, some of them showing homozygous lethality. To exclude the possibility that their lethality was caused by background mutations, the lethal alleles were crossed to the Df(3R)Exe16269 specific deletion in complementation tests. Further molecular analysis revealed that four of these mutants (5%) are short deletions, two of them removing only the 5¢ part of the DmManf gene while the other two partly deleting both DmManf and CG14879 neighboring genes, flanking the original P{EP} element insertion. In homozygous and trans-heterozygous combinations, these mutations showed polyphasic lethality. These results prove that that affected genes are important for normal development and essential for viability. The mutants we recovered are essential for a detailed analysis of these genes functions, especially for DmManf, which is the fruit fly ortholog of the human Manf gene, a possible oncogene. Acknowledgements: This work was supported by CNCSISUEFISCDI, PNII- IDEAS grant no.1004/2009, code 1936/2008.


YSF – Young Scientist Forum YSF.64 Subcellular localization and structural properties of tobacco 4/1 protein S. S. Makarova1, T. N. Erokhina2, A. G. Solovyev3, J. Schiemann4, R. Owens5 and S. Y. Morozov3 1 Department of Virology, Moscow State University, Moscow, Russia, 2Department of Molecular Diagnostics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia, 3 Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia, 4Institute for Biosafety of Genetically Modified Plants, Julius Kuehn Institute (JKI), Quedlinburg, Germany, 5Molecular Plant Pathology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, USA At-4/1 protein has been identified as an Arabidopsis thaliana polypeptide of unknown function capable of interaction with a plant virus movement protein involved in cell-to-cell transport of viral infection. At-4/1 protein has been shown to accumulate in the cytoplasm of plant cells in a polar fashion and to move through plasmodesmata to neghbouring cells. Here we report isolation of Nt-4/1 gene, the 4/1 gene from tobacco (Nicotiana tabacum), and subcelullar localization of the encoded protein as well as its structural properties. The Nt-4/1 protein fused to GFP, as it was previously described for At-4/1-GFP fusion protein, localizes in numerous small vesicular structures dispersed throughout the cytoplasm and, in addition, in the cell nucleus. Dynamic laser light scattering analysis revealed that the bacterially expressed Nt-4/1 protein can form dimers and trimers in vitro. Analysis of circular dichroism spectra of the Nt-4/1 protein demonstrated that it has a pronounced a-helical secondary structure. Using differential scanning calorimetry we revealed at least three calorimetric domains in the Nt-4/1 molecule.

YSF.65 Myocardial oxidative stress in an experimental model of nonalcoholic fatty liver disease: role of silibinin M. Malaguarnera, F. Salamone2, F. La Delia1, F. Galvano1 and G. Li Volti1 1 Department of Drug Sciences, Section of Biochemistry, University of Catania, Catania, Italy, 2Department of Internal Medicine, University of Catania, Catania, Italy Non Alcoholic Fatty Liver Disease (NAFLD) is a chronic liver disease with a significant impact on cardiovascular morbility/ mortality. Oxidative stress, lipotoxicity and inflammation are considered to play a major role in the pathogenesis of liver and heart injury in NAFLD. Silibinin, a flavonolignan extracted from milk thistle, exerts a marked protective action in a variety of experimental liver injuries. We aimed at identifying silibinin effects in the liver and in the myocardial tissue of db/db mice fed a methionine choline deficient (MCD) diet. We explored the effects of a 4-week daily (20 mg/kg i.p.) administration of silibinin in db/db mice fed a MCD diet. Insulin resistance was evaluated by HOMA-IR. Liver and myocardial histology was assessed by standard procedures; ultrastructural morphology was evaluated by electron microscopy. Liver and heart triglycerides and free fatty acids (FFA) were determined by colorimetric kits; apoptosis was assessed by the TUNEL assay. Isoprostanes and 8-hydroxyguanosine (8-OHG) were determined by ELISA; reduced glutathione (GSH) was assessed by a GSH assay; nitrite/ nitrates were measured colorimetrically; TNF-a and IL-6 were determined by ELISA. Silibinin administration decreased HOMAIR, markedly improved liver and myocardial histology, decreased liver and heart triglycerides and FFA; ultrastructural

Abstracts mitochondrial morphology was preserved in both tissues; the number of apoptotic cells in the liver and heart of treated db/db mice was similar to the levels observed in lean animals. Silibinin treatment strongly decreased liver and heart isoprostanes, 8OHG, nitrite/nitrates and increased GSH. Consistently, silibinin completely restored the levels of TNF-a and IL-6 in both organs. In this study we demonstrated that silibinin exerts potent hepatoprotective and cardioprotective effects in obese diabetic mice. These findings may provide a strong rationale for the use of silibinin in the management of patients with NAFLD.

YSF.66 Novel phosphatase for tumor suppressor TSC2? O. M. Malanchuk, S. S. Palchevskyy and V. V. Filonenko Department of Cell Signaling, The Institute of Molecular Biology and Genetics of NASU, Kyiv, Ukraine The signalling components of the protein kinase mammalian target of rapamycin (mTOR) pathway are frequently altered in a wide variety of human diseases. Upstream of mTOR key signalling molecules are the small GTPase Ras, the lipid kinase PI3K, PKB, and the GTPase Rheb, which are known to be deregulated in many human cancers. Mutations in the mTOR downstream pathway component genes TSC1 or TSC2 trigger the development of the Tuberous sclerosis syndrome (TSC), connected with the development of hamartomas in numerous organ systems. Both protein products of TSC1 and TSC2 genes form an intracellular complex. Recent progress in understanding the mechanisms of growth control indicates that the tumour-suppressor tuberous sclerosis complex TSC1/2 plays a major role in negative regulation of cell growth. Tumour-suppressor tuberous sclerosis complex TSC1/2 exerts GAP activity towards a small GTP binding protein Rheb. The activity of TSC1/2 complex is regulated by multiple phosphorylations of TSC2 mediated by several kinases, such as PKB, AMPK, etc. So far, very little is known about the molecular mechanisms of TSC2 dephosphorylation. In our previous research using the Y2H-screening we have identified a number of potential TSC2 binding partners including protein phosphatase 5 (PP5). The aim of this study was to characterize interactions between TSC2 and PP5. Data presented in this work indicate that TSC2 interacts with PP5 in vivo and efficiency of such interaction depends on physiological status of cells. In addition, PP5 is capable of dephosphorylating TSC2 in vitro at AMPK potential sites. Our data suggest that the physiological relevance of TSC2/PP5 interaction should be in reversing the stimulatory signaling mediated by AMPK on TSC2 activity.

YSF.67 Extracellular matrix nano-mechanics determines megakaryocyte function A. Malara1, C. Gruppi1, V. Abbonante1, E. Spedden2, R. Tenni1, M. Raspanti3, D. Kaplan4, M. E. Tira1, C. Staii2 and A. Balduini1,4 1 Biotechnology Laboratory, Department of Biochemistry, IRCCS San Matteo Foundation, University of Pavia, Pavia, Italy, 2 Department of Physics, Tufts University, Medford, MA, USA, 3 Department of Human Morphology, University of Insubria, Varese, Italy, 4Department of Biomedical Engineering, Tufts University, Medford, MA, USA Cell contact with extracellular matrix proteins (ECMs) leads to activation of specific biochemical signaling pathways and to cyto-


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Abstracts skeletal modifications that regulate processes such as cell differentiation, migration and apoptosis. Mechanical properties of ECMs play an important role in determining cells behavior during these processes. In the bone marrow, ECMs concur to the generation of cues that are important for hemopoietic stem cells maturation and differentiation. Sites around the endosteal bone and vascular districts have been proposed as critical niches for stem cell differentiation into megakaryocytes (Mks). In this scenario, fibrillar type I collagen seems to be a key regulator of platelet release, as in vitro adhesion of Mks to this protein inhibits platelet release through the generation of a bulk cell contraction mediated by mechano-sensitive proteins, such as fibronectin, Rho-GTPase and myosin, that lead to cell spreading overtime. In this work we have used a chemical modified collagen that completely override in vitro collagen ligand pathways in directing Mks response in term of cell spreading, migration, platelet release and fibronectin assembly. This different behavior seems to be related to the different nano-mechanical properties of modified collagen with respect to native protein. In particular, N-acetylation of lysine side chains of collagen blocks the formation of banded fibrils and self-aggregation leading to differences in the in vitro sopramolecular organization. Atomic force microscopy analysis of Mks interaction with collagens clearly demonstrated that absence of fibrils, despite similar integrin engagement, and different mechanical properties of these proteins, regulate Mks behaviour and fate. New insights into signaling pathways and in mechano-sensing systems of cells need to be addressed but nanoscale mechanical properties of ECMs seem to have an important role in regulating megakaryocyte behavior in vitro and probably in vivo.

YSF.68 EDEM1 interacts with misfolded tyrosinase mutants in a glycan independent manner M. Marin1, S. Ghenea1, G. Chiritoiu1, L. Spiridon2, A.-J. Petrescu2 and S. M. Petrescu1 1 Molecular Cell Biology Department, Institute of Biochemistry, Bucharest, Romania, 2Bioinformatics and Structural Biochemistry Department, Institute of Biochemistry, Bucharest, Romania Non-native and immature proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery and degraded by ERAD (ER-associated protein degradation). EDEM (ER degradation-enhancing a-mannosidase like protein) proteins are ERAD components, in the proximity of the export channel, presumed to guide the disposal of misfolded glycoproteins from the ER. EDEM1 was shown to interact with terminally misfolded proteins from the ER, possibly targeting them for retrotranslocation and proteasomal degradation. It has been postulated that the other two EDEM proteins are also involved in protein targeting to degradation, but the mechanism for all three proteins it not yet clear. Our aim is to understand the molecular mechanism that discriminates between native and malfolded proteins, investigate the association of the ERAD substrates with EDEM proteins and establish their role in this pathway. In order to determine the interaction of EDEM1 with misfolded proteins we used as model ERAD substrate human tyrosinase mutants. We obtained several glycosylation tyosinase mutants and few EDEM1 mutants in order to determine if the interaction between the two proteins is lectin type or not. Experimental data showed that EDEM1 interacts with both folded and misfolded ERAD substrates, in native and also denaturant conditions, showed by pulse-chase and immunoprecipita-

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YSF – Young Scientist Forum tion methods and it is also an interaction partner for SEL1L, a component of the retrotranslocon in eukaryotes.

YSF.69 Novel derivatives of cationic lipids for delivery of DNA and RNA into dendritic cells O. V. Markov1, M. A. Maslov2, N. L. Mironova1, G. A. Serebrennikova2, V. V. Vlassov 1 and M. A. Zenkova1 1 Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia, 2Lomonosov Moscow State Academy of Fine Chemical Technology, Moscow, Russia Triggering of antitumor immune response by ex vivo generated dendritic cells (DC) pulsed with tumor-associated antigens (TAA) is the perspective approach to enhance the efficiency of anticancer therapy. One of the main impediments in this therapy is poor uptake of nucleic acids encoding TAA by DC. We studied the ability of liposome built of a novel cholesterol based cationic lipids and lipid-helper DOPE (dioleoylphosphatidylethanolamine) to deliver DNA (plasmid pEGFP encoding enhanced green fluorescent protein) and RNA (total RNA isolated from BHK-IR780 cells expressing endogenously EGFP) into murine immature DC and their bone marrow progenitors. Cationic lipids contain one (X2, S1, S2 and S3) or two (2X3) cholesterol residues or long-chain hydrocarbon substituent (2D3) linked with spermidine. DC progenitors were obtained by density gradient centrifugation. Immature DCs were isolated by immunoadsorption of CD34+ DC progenitors from bone marrow homogenate followed by cell culturing in the presence of GMCSF and IL-4 for 6 days. Data show that lipids 2X3 and S2 display high efficiency in the cellular delivery of plasmid DNA: 23% of DC progenitors and 15% of immature DC were transfected pEGFP with fluorescence intensities 4.5–13 and 5–5.2 RFU (relative fluorescent units), respectively. Lipids 2X3, 2D3 and X2 were the most effective in the delivery of total RNA: 13–15% of cells were transfected and fluorescence intensity was 1.4–4.9 RFU. The studied lipids exhibited much higher efficiency of DNA and RNA delivery as compared to Lipofectamine. The obtained data show that the novel cholesterol based cationic liposome can be applied for efficient transfection of DC with nucleic acids (DNA or RNA) encoding TAA thus enhancing their immunogenicity. This work was supported by RAS programs ‘‘Molecular and cellular biology’’ (No. 22-1), ‘‘Fundamental sciences to medicine’’ (No. 21-19), Interdisciplinary grant of SB RAS (No. 15) and Scientific School – 7101.2010.4.

YSF.70 Chitosan-TPP/siRNA mediated Hsp70 knockdown: potential cancer therapeutic M. Matokanovic, K. Barisˇ ic, A. Hafner and J. Filipovic-Grcic Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia,Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Apoptosis resistance of tumor cells is associated with increased expression of heat shock proteins (Hsp). RNA interference nanotechnology can be used for Hsp gene silencing, in order to accomplish greater sensitivity of tumor cells against chemotherapeutics, which are based on apoptosis promotion. Solution of chitosan, in the form of glutamate salt (75% stenosis in one and three and more coronary arteries respectively) was assessed. Results showed that rs1333049 risk allele C was associated with three vessel disease (OR = 1.45, p = 0.01, adjusted for genre, age, smoking status and BMI) however no association was found with one vessel disease or number of coronary arteries with severe stenosis. Genre stratified analysis showed that allele C is associated with three vessel disease in men (n = 392, OR = 1.71, p = 0.001, adjusted for genre, age, smoking status and BMI) but not in women (n = 100, OR = 1.27, p = 0.47). Conclusion: Common SNP risk allele predicts severity of coronary artery disease in men, but not women.

Abstracts YSF.91 (S5.2.6) Rab GTPases and Mast Cell Exocytosis N. Pereg-Azouz, Z. Naomi2, F. Mitsunori3 and S.-E. Ronit1 1 Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 2The Biophysical Interdisciplinary Schottenstein Center for the Research and the Technology of the Cellome, Department of Physics, Bar Ilan University, Ramat Gan, Israel,3Fukuda Initiative Research Unit, RIKEN, Wako, Saitama, Japan Mast cells (MC) are specialized secretory cells involved in innate and adaptive immune responses as well as in mediating allergic and inflammatory reactions. These cells are packed with secretory granules (SG) which contain allergic, inflammatory and immunoregulatory mediators. When triggered, multiple signaling events are activated resulting in fusion of the SGs with the plasma membrane (degranulation). Efforts are being undertaken to develop novel therapies that will specifically target MC activation. Adopting screening approaches aiming to unveil stimulus-secretion coupling networks in MC has been limited due to their low transfection efficiency. Hence, genetic manipulations are unlikely to leave an impact on the actual readouts of average secretion measured by conventional methodologies. We established a technology that allows functional and phenotypic-driven screens. Using this technology, we have screened the Rab family of small GTPases. Rab proteins localize to distinct membrane-bound compartments, where they regulate transport and fusion events. Therefore, identification of Rab network that controls MC exocytosis should unveil the cellular machineries involved in the execution and regulation of this process. We have already identified 22 Rabs as significant modulators of MC degranulation. To our knowledge the involvement of 18 proteins, out of this list was heretofore not recognized. Rabs that serve as positive regulators might serve as cellular targets for future developments of therapeutic agents to treat allergic and inflammatory diseases. We are further analyzing the pattern of individual cell responses to manipulations of the relevant Rabs under defined cellular settings by using a chip-based nano-system. We are combining multidisciplinary approaches including cell and molecular biology, and computational techniques aiming to elucidate the mechanisms by which these proteins affect this process thus deciphering the Rab networks that regulate exocytosis

YSF.92 GSK3-SCFFbw7 targets junB for degradation in G2 to preserve chromatid cohesion B. Perez-Benavente1, J. L. Garcia2, M. S. Rodriguez3, J. F. de Mora1 and R. Farras1 1 Prince Felipe Research Centre (CIPF), Av. Autopista del Saler 16, Camino de las Moreras, Valencia, Spain, 2Unidad de Investigacion, Instituto Estudios Ciencias de la Salud de Castilla y Leon (IECSCYL)-Hospital Universitario de Salamanca, Centro de Investigacion del Cancer Universidad de Salamanca-CSIC, Salamanca, Spain,3The proteomics Unit, CIC BioGUNE, CIBERehd, Derio, Bizkaia, Spain JunB, a member of the AP-1 transcription factor, is a cell context-dependent tumour supressor or oncogen. Overexpression of JunB has been linked to lymphoma pathogenesis. However, the role of JunB in promoting neoplastic growth is not fully understood. JunB undergoes coordinated phosphorylation-dependent ubiquitylation in G2. We have found that a critical consensus phosphodegron within the JunB sequence controls its turnover and we identified GSK3 and SCFFbw7 as, respectively, the kinase and the E3 ubiquitin ligase responsible for efficient JunB degradation in G2. Notably, pharmacological or genetic inactiva-


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Abstracts tion of the JunB-GSK3-Fbw7 degradation module induced accumulation of JunB in G2/M and produced transcriptional repression of DDX11, a DNA helicase that regulates sister chromatid cohesion. Cells with repressed levels of DDX11 displayed deficient chromatid cohesion. Our results reveal a novel mechanism by which GSK3/SCFFbw7-mediated proteolysis controls mitosis progression and chromatid cohesion by degrading JunB, an essential step in maintaining genetic fidelity through mitosis.

YSF.93 Human galectin-1 interacts with porphyrins L. Petrova, G. Russev, C. John, A. Varriale, S. D‘Auria and V. Bogoeva Institute of Molecular Biology, Bulgarian Academy of Sciences Lectins are carbohydrate-binding proteins, known for their ability to interact with specific oligosaccharides. Some animal lectins have non-carbohydrate binding domains, and it is shown that they bind non-carbohydrate ligands through hydrophobic interactions. Therefore, we postulated that human lectins, in particular galectin-1, could interact with non-carbohydrate ligands. The present work shows that the tumor specific lectin – human galectin-1 (hGal-1) interacts with three porphyrin compounds: Zn-, Mnand Au-porphyrins. The interaction of ZnTPPS with hGal-1 was studied by the specific fluorescence emission of the porphyrin. The protein binding properties to Mn/Au porphyrins were measured by intrinsic protein fluorescence quenching. The values determined for the apparent dissociation constants (KD) of 0.6– 1.5 lM are similar to the KD for complexes of Concanavalin A and porphyrin, and are indicative of the high affinity of hGal-1 for these porphyrins. In addition, the analysis of the hyperbolic binding curves suggests the presence of one hGal-1 binding site for porphyrins. We also found that hGal-1 interacted with the fluorescent probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) that was used to identify the hydrophobic regions within hGal-1. Our data indicate that hGal-1 similar to some plant lectins, a bacterial lectin from Pseudomonas aeruginosa and an animal lectin from Helix pomatia possesses dual functions binding to both carbohydrate and non-carbohydrate ligands. In summary hGal-1 shows a novel porphyrin binding activity, in addition to its carbohydrate one. Regarding this, hGal-1 could be viewed as a potentially interesting delivery molecule for the treatment of cancer cells.

YSF.94 Towards the structural characterization of the MobM relaxase in complex with its cognate DNA. Insights into the DNA transfer mechanism of gram-positive bacteria R. Pluta1, S. Russi1, F. Lorenzo2, R. Pe´rez-Luque1, R. Boer1, M. Espinosa2 and M. Coll1 1 Institute for Research in Biomedicine (IRB Barcelona) and Institut de Biologia Molecular de Barcelona (CSIC), Barcelona, Spain, 2Centro de Investigaciones Biolo´gicas (CSIC), Campus de la Ciudad Universitaria de Madrid, Madrid, Spain Bacterial conjugation is a major mechanism of the horizontal gene transfer in bacteria and thus an important component of bacterial evolution. It provides a route for the rapid acquisition of new genetic information and contributes to the spread of anti-

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YSF – Young Scientist Forum biotic resistance. The latter is becoming a serious problem. Currently, 70% of all hospital acquired infections are resistant to at least one antibiotic, and antibiotic resistance to at least one drug has been reported for every major strain of pathogenic bacteria. Despite that, the horizontal DNA transfer in Gram-positive bacteria is poorly understood at the molecular level. We are investigating the MobM protein from the broad-host range plasmid pMV158 of Gram+ bacteria Streptococcus agalactia. MobM is a protein of the relaxase family that initiates DNA replication and transfer. MobM and other super-family pMV158 relaxases specifically bind a 6–10 bases long loop formed by an inverted repeat sequence (oriT) and cut the plasmid DNA at the nic site downstream of the loop. This allows separation of one DNA strand and its passage through Type IV Secretion System to the other bacteria. Co-crystals of the complex between different MobM variants and oligonucleotides that mimic the oriT sequence just upstream the nic site are being prepared and initial crystals of the N-terminal relaxase domain in complex with 26 bases long DNA are under optimization process.

YSF.95 (S5.3.6) Quantitative proteomics analysis of secretome and secreted microvesicles of chronic myeloid leukemia cells using SILAC method P. Podszywalow-Bartnicka1, M. Tkaczyk2, M. Dadlez2 and K. Piwocka1 1 Nencki Institute of Experimental Biology, Polish Academy of Science, Warsaw, Poland, 2Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML) and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. As a model of CML, we employed mouse progenitor 32D cells expressing high Bcr-Abl level corresponding to blast crisis cells as well as human K562 cells. There is a growing interest in determining the role of the microenvironment in regulating growth, self-renewal and drug resistance of leukemic stem cells. Communication of leukemic progenitor and stroma cells might play a crucial role in leukemia progression. Secretome enabling intercellular signal transduction corresponds to either soluble proteins secretion or microvesicles exocytosis, what bypass the information or regulatory signal by the usage of both integrated proteins and microRNA. Signaling pathways are commonly deranged in cancer and quantitative proteomics offers powerful approaches to map these pathways and their aberrations in cancer. Stable isotope labeling with amino acids in cell culture (SILAC) is one of the most used tools for quantitative comparison of protein abundance which uses the light and heavy peaks observed in the mass spectrum. By this approach we identified soluble extracellular proteins secreted by K562 human CML cell line. Moreover, we compared protein abundance in secreted microvesicles (exosomes enriched fraction) of 32D cells and Bcr-Abl expressing cells. Most of the proteins found in microvesicles from CML cells were not present in 32D exosomes, showing Bcr-Abl-dependent regulation. Most of the proteins identified in the CML microvesicles were involved in the regulation of cell movement, extracellular matrix function, signal transduction, viability and others. Also new proteins were identified. This might shed light on the importance of the stroma-leukemic progenitor cells cross talk and deliver new terapeutic targets as well as diagnostic marker.


YSF – Young Scientist Forum YSF.96 In vitro studies of EPM1 mutants of human stefin B M. Polajnar, N. Kopitar-Jerala, V. Turk and E. Zerovnik Department of Biochemistry, Molecular and Structural Biology Human stefin B is the most labile and amyloidogenic protein among the members of the broader family of cystatins. Its prefibrillar oligomers showed to be toxic in in vitro and ex vivo studies (Cˇeru et al., 2008). Mutations in the gene of stefin B are responsible for the primary defect underlying EPM1. EPM1 is a rare progressive myoclonic epilepsy with generalized tonic-clonic seizures and slow progressive mental deterioration. Mutants by majority cause lower expression or loss of protease inhibitory activity, which would implicate lack of protein’s protease function as the most likely cause. However, the physiological function of stefin B is still under investigation. The lack of stefin B increases apoptosis and oxidative stress in affected neurons, implying it has a protective role in the brain. The protein is also over-expressed in status epilepticus and after seizures, which could potentially cause its aggregation. It was predicted that stefin B as well as some EPM1 mutants might aggregate in cells (upon over-expression) and thus gain in ‘‘toxic function’’ (Cˇeru et al., 2005). Some pathological mutants of stefin B observed in EPM1 patients have been prepared and their stability and aggregation studied (Rabzelj et al., 2005). Here we report on some new EPM1 mutants of stefin B: G50E and Q71P. Studies of their solution structure showed that they are partially unfolded and form a molten globule. This was confirmed by ANS fluorescence, which showed a large amount of exposed hydrophobic surfaces. No denaturation studies could be performed due to their instability. Contrary to WT and G4R mutant and the R68X fragment, these two mutant proteins (G50E and Q71P) do not form fibrils at acidic pH but rather non-amyloid amorphic aggregates.

YSF.97 RAGE maturation and processing in melanoma cells I. Popa, E. Ganea and S. Petrescu Institute of Biochemistry, Bucharest, Romania RAGE is a cell surface signaling receptor for different types of ligands, such as advanced glycation end products, amphoterin, members of S100/calgranulin family, amyloid peptides and bfibrils. RAGE expression is up-regulated at sites where its ligands accumulate, promoting a sustained cellular activation which may lead to pathological responses in inflammation, cancer, diabetes and amyloidosis. Different structural regions of the extracellular domain of RAGE provide anchorage sites for different ligands, determining certain signaling cascades to be turned on. An extra layer of specificity of RAGE activation is acquired through N-glycosylation. Data in literature show that modifications by carboxylated N-glycans on RAGE mediate ligand binding, receptor oligomerization, and subsequent cellular signaling. Such interactions would be important in inflammatory mediated carcinogenesis. Moreover, a multitude of RAGE isoforms is generated by alternative splicing or proteolytical cleavage, in a tissue or cell-type specific manner. It has been already suggested a role of RAGE in tumor growth and metastasis, however modulation of RAGE expression and localization in connection with tumor progression has not been thoroughly addressed. To add more knowledge in this field we used radioactive labeling and pulse chase experiments, subcellular fractionation on density gradients, Western blot, immunofluorescence and secretion experiments in cultured melanoma cells. Our

Abstracts results present new data on RAGE synthesis, maturation to the N-glycosylated form, oligomerization, intracellular localization, and secretion of RAGE by melanoma cells with different degrees of malignancy.

YSF.98 Structural characterisation of an ubiquitin hydrolase: AMSH (associated molecule of the SH3 domain of STAM) E. Poudevigne, J. Solomons and W. Weissenhorn Unit of Virus Host Cell Interactions (UVHCI) UMI 3265 Universite´ Joseph Fourier-EMBL-CNRS, Grenoble Cedex 9, France ESCRT (Endosomal Sorting Complexes Required for Transport) proteins trigger the budding of biological membranes away from the cytosol in processes such as virus budding, degradation of receptors via the endosomal pathway, cytokinesis or autophagy. These mechanisms need the interaction with ubiquitin but also deubiquitin enzymes such as UBPY (UBiquitin- specific processing Protease Y) or AMSH (Associated Molecule of the SH3 domain of STAM). Further biochemical and structural knowledge about AMSH will help to understand the regulation of ESCRT proteins. In particular, we use crystallization, Surface Plasmon Resonance, pull-downs and other biophysical and biochemical techniques in order to characterize the AMSH protein and its binding partners; among them, the ESCRT-III CHMP (Charged MVB Protein) proteins are responsible for membrane deformation and scission. A crystal structure, SPR data and colocalization in mammalian cell have been obtained for AMSH in complex with CHMP3 in our previous work. Next we will focus on the interaction between AMSH and other CHMP proteins, notably CHMP1A and 1B and we will try to characterize the active form of this ubiquitin hydrolase. The results will allow us to better understand the regulation of ESCRT-III proteins and thus the membrane deformation and scission processes.

YSF.99 Interplay between two upstream open reading frames (uORFs): role in stress- induced translational regulation H. Raveh-Amit, J. Poller and E. Livneh Department of Microbiology and Immunology, Ben Gurion University of the Negev During nutrient deprivation, eukaryotic cells need to synthesize proteins essential for coping with the stress in the face of a limited supply. This requirement is achieved by the phosphorylation of eukaryotic initiation factor 2 that leads to a reduction in global protein translation accompanied by translational upregulation of proteins needed for the stress response. We showed that the translation of a Protein Kinase C (PKC) isoform, PKCeta, is upregulated following nutrient deprivation by two conserved upstream Open Reading Frames (uORF1 and uORF2) in its 5¢ Untranslated Region [Raveh-Amit et al. Mol Cell Biol 2009]. Here, we examined the translational control mechanism by these uORFs during stress using mutagenesis analysis in a luciferase reporter plasmid containing the 5¢ Untranlated Region of PKCeta. First, we examined reinitiation at the main ORF following translation of uORF2. A frame shift mutation in uORF2 that abrogated reinitiation did not alter the luciferase activity, indicating that reinitiation at the main ORF does not normally occur following translation of uORF2. Furthermore, following nutrient starvation the induction of the main ORF was not hampered by this mutation, providing evidence that ribosomes bypass uORF2


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Abstracts under stress. Secondly, we evaluated the effect of uORF1 on leaky scanning of uORF2. A mutation in the initiation codon of uORF1 idecreased the reporter activity, indicating that reinitiation at the main ORF following translation of uORF2 becomes possible when uORF1 is bypassed. Furthermore, this mutation decreased the reporter activity under stress conditions, supporting that ribos omes bypass uORF2 under these conditions regardless of uORF1. Altogether, our studies show that PKCeta is regulated by mechanisms of reinitiation and leaky scanning of the two uORFs and shed new light on translational control by uORFs in response to cellular stress.

YSF.100 Cancer stem cells and dedifferentiation: implications in lung cancer C. F. D. Rodrigues1,2,3,4, C. M. F. Gomes4,5, A. D. Sampaio1,3,4, M. F. Botelho4,6, A. M. Abrantes4,6, L. Carvalho4,6, A. B. Sarmento4,6, R. Carvalho1,3, I. M. Carreira4,6 and M. C. Alpoim1,3,4 1 Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 2PhD Programme on Biomedicine and Experimental Biology, CNC, University of Coimbra, Coimbra, Portugal, 3Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal, 4Centre for Research in Genetics, Environment and Oncobiologia, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 5 Biomedical Institute for Light and Image Research, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 6Faculty of Medicine, University of Coimbra, Coimbra, Portugal In the last decades cancer has evolved as the most common human disease with lung cancer toping the ranking. All over the world different theories have tried to explain the emergence, development and progression of human tumors, although none have yet fully succeeded on its task. One of the recent and most appealing theories argues that the heterogeneous nature of each tumor encompasses a specific population of cells, with Stem Cells (SCs) like properties, which gives rise to the bulk of the tumor cells with more differentiated phenotypes (Lobo et al., 2007). Although these Cancer Stem Cells (CSCs) have been blamed as responsible for tumor recurrence and resistance to conventional therapies (Kitamura et al., 2009), their origin is rather controversial. Aiming to understand the mechanisms underlying hexavalent chromium [Cr(VI)] induced lung cancer, we succeeded to induce the malignant transformation of the non-tumorigenic bronchial epithelial cell line (BEAS-2B) following exposure to occupational relevant Cr(VI) concentrations (Rodrigues et al., 2009). Subsequently, the resulting RenG2 cell line malignant potential was increased following successive rounds of transplantations in athymic nude mice (submitted manuscript). Metabolic and cell cycle studies revealed that the more malignant RenG2-derivative cell lines (DRenG2 and DDRenG2) have increased glycolytic metabolism and decreased replicative index. Additionally, sphere-formation assays also revealed positive only for DRenG2 and DDRenG2 cell lines and the molecular characterization of those cultures confirmed their CSCs-identity. Apparently, the increased malignant potential of DRenG2 and DDRenG2 cell lines can be ascribed to a process of cellular dedifferentiation leading to the emergence of CSC-like sub-populations in both cell lines boosting their aggressiveness and resistance.

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YSF – Young Scientist Forum YSF.101 Protein dysfunction in mitochondrial fatty acid beta oxidation accounts for oxidative stress burden J. V. Rodrigues1, B. J. Henriques1, R. Olsen2, P. Bross2 and C. M. Gomes1 1 Instituto Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal, 2Research Unit for Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark Defects in the mitochondrial electron transfer flavoprotein (ETF) account for multiple acyl-CoA dehydrogenation deficiency (MADD), a disorder of fatty acid, amino acid and choline metabolism. Appearance of MADD symptoms associated with the mild phenotype is often determined by sporadic factors which remain largely unknown. In this respect, oxidative stress may be an important triggering factor for the development of MADD. Although it has been postulated that ETF could be a source of reactive oxygen species (ROS), no quantitative measurements have been performed so far. Here, we employ a newly developed methodology (1) to quantify superoxide and hydrogen peroxide produced by human ETF and ETF mutants that have been identified in MADD patients. We found that mutations which yield dysfunctional ETF proteins (2,3) lead to increased oxidative stress. In addition, we have in vitro evidence for a striking role of fatty acid metabolism pathway enzymes in controlling the extent of oxidative stress. The implications of these results on the overall ROS produced by mitochondrial metabolism, as well as its possible impact in MADD are discussed. References: 1. Rodrigues J. V., and Gomes C. M. Free Radic Biol Med 2010; 49 (1): 61–66. 2. Henriques B. J., Rodrigues J. V., Olsen R. K., Bross P., and Gomes C. M. J Biol Chem 2009; 284 (7): 4222–4229. 3. Henriques B. J., Bross P., and Gomes C. M. Biochim Biophys Acta 2010; 1802 (11), 1070–1077.

YSF.102 From in silico to in vitro substrate characterization of Macaca fascicularis P450 2C20 F. Rua, S. J. Sadeghi and G. Gilardi Department of Human and Animal Biology, University of Turin, Turin, Italy Macaques are the most widely used nonhuman primates in preclinical studies to obtain FDA approval of new drugs but the lack of information on cytochrome P450 genes has hampered the understanding of drug metabolism in this species. Human P450 2C8 metabolizes more than 8% of drugs cleared by Phase I reaction but today there are no data on the substrates recognized by the homologous Macaca fascicularis P450 2C20. In this work, a 3D model of cynomolgus 2C20 was generated and optimized on the basis of the available crystal structure of the P450 2C8 by employing the YASARA program. A set of 60 known substrates of P450 2C8 were taken from the literature and docked in the active site of both the 2C20 model and the available 2C8 structure. Based on the predicted binding energies, both proteins can potentially bind the selected P450 2C8 substrates with similar affinity. In order to validate the in silico docking data by in vitro methods, the gene encoding CYP2C20 was cloned in pCW vector and successfully expressed in E. coli. Furthermore, a chimeric protein consisting of P450 2C20 and the soluble reductase domain of CYP102A1 (Bacillus megaterium-BMR) was engineered. The solubility and catalytic self-sufficiency of this protein would greatly


YSF – Young Scientist Forum simplify the in vitro preclinical studies of cytochrome P450 2C20 mediated drug metabolism. Three drugs were then selected from the energy output profile of the in silico docking experiments namely Paclitaxel, Torzasertib (anti-cancers) and Amodiaquine (anti-malarial) for in vitro testing with the purified P450 2C20 and its chimera. The turnover of the P450 2C20 and the chimera led to Km values of 1.9 ± 0.2 and 5.9 ± 2.3 lM for Paclitaxel; 1.5 ± 0.2 and 2.1 ± 0.7 lM for Tozasertib; 1.2 ± 0.2 and 1.6 ± 0.17 lM for Amodiaquine, respectively. Finally, the data obtained from the in silico and in vitro analysis demonstrate the ability of the cynomolgus P450 2C20 to recognize and turnover P450 2C8 substrates, its human homologue.

YSF.103 A novel perspective in the metabolism of glutathione: antioxidant and antioapoptotic function of c-glutamilcysteine in mitochondria R. Quintana-Cabrera, A. Almeida and J. P. Bolanõs Laboratorio de Bioquı´mica y Biologıá Molecular de la Neurodegeneracioń, Instituto de Neurociencias de Castilla y Leoń, Universidad de Salamanca, Salamanca, Spain Mitochondria are the main source of reactive oxigen species (ROS) in the cell, being also the most important target of their effects. Although the exact mechanisms remain elusive, mitochondrial ROS can regulate different signal pathways through the interaction with mitochondrial proteins. In this work, our aim was to design and characterize a system capable of downregulating the formation of ROS specifically in the mitochondria. For this purpose, the mitochondrial-targeting domain of ornithine transcarbamylase was fused to the N-terminal domain of glutamate-cysteine ligase, catalytic subunit (GCLh). This mito-GCLh cDNA was expressed in neurons and HEK293T cells, which resulted in the efficient targeting of GCLh to the mitochondria, as confirmed by subcellular fractionation and western blotting. The protein was proved to be functional within the organelle, as assessed by c-glutamylcysteine formation in isolated mitochondria, measured by HPLC with electrochemical detection. The production of H2O2 and O2·) detected with the Amplex Red and MitoSOX probes respectively, was found to be lower in mitochondria isolated from cells expressing mito-GCLh when compared with those expressing cytosolic GCLh. Mito-GCLh also supported protection against excitotoxic damage-mediated caspase 3 activation and apoptotic cell death. These effects could be wholly accounted for by the presence of GCLh in the mitochondria, being fully independent on the formation of glutathione in the cytosol, which did not differ between controls and mitoGCLh expressing cells. In conclusion, our results show that cglutamylcysteine can act as an antioxidant that downregulates the formation of ROS specifically in the mitochondria. This strategy represents a novel tool for the study of signaling pathways modulated by mitochondrial ROS as well as a new defense system against oxidative stress.

Abstracts YSF.104 New mouse models to study HGF/Met role in postnatal heart development and heart disease pathogenesis V. Sala1, C. Leo1, S. Gallo1, S. Gatti1, M. Morello2, A. Chiribiri2, E. Bucci3, E. Medico4, D. Cantarella4, I. Riess1, S. Schiaffino5, D. Mancardi6, C. Ponzetto1 and T. Crepaldi1 1 Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Italy, 2Department of Internal Medicine, University of Turin, Italy, 3BioDigitalValley, Pont Saint Martin, AO, Italy, 4Institute for Cancer Research and Treatment, University of Turin, Italy, 5Venetian Institute of Molecular Medicine (VIMM), Padoa, Italy, 6Department of Clinical and Biological Sciences, University of Turin, Italy The Hepatocyte Growth Factor (HGF) is involved in many physiological processes, including heart development. In the adult damaged heart, HGF exerts cardioprotective, antiapoptotic and regenerative effects. We aimed to explore the role of HGF/Met receptor in cardiac postnatal development and heart disease. To this purpose, we generated two transgenic mouse models, with drug-inducible and cardiac-specific expression of HGF or TprMet, the activated form of Met. HGF overexpression caused reduced Connexin43 (Cx43) and sarcomeric proteins in newborns, as respect to controls. We performed full-genome microarray analysis of mouse hearts at postnatal days 2 and 7, when the transition between plastic and post-plastic phases occurs. At P7, we found 249 genes significantly modulated in HGF-expressing hearts. Most of the genes upregulated by HGF were downregulated from P2 to P7 in control hearts, suggesting that HGF opposes to physiological downregulation. We confirmed that HGF increases proliferation at P7, indicating that it prolongs the plastic phase in the neonatal heart. As adults, mice overexpressing HGF developed a systolic contractile dysfunction, even when HGF expression was suppressed after birth. Interestingly, levels of Cx43 were maintained low throughout the lifespan. Prenatal expression of Tpr-Met was lethal soon after birth. When inducing Tpr-Met transgene starting from postnatal life, we generated a model of congestive heart failure, characterized by reduced Cx43, upregulation of foetal genes, concentric hypertrophy, diastolic dysfunction, with altered ECG and signs of cachexia. These data suggest that HGF/c-Met axis exerts an important role in heart postnatal development and might be implicated in the pathogenesis of cardiac disease with developmental origins.

YSF.105 Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex R. Sanjuan-Jimeńez1, J. D. Colmenero2 and P. Morata1 1 Biochemistry and Molecular Biology Department, Faculty of Medicine, University of Malaga, Malaga, Spain, 2Infectious Diseases Service, Carlos Haya University Hospital, Malaga, Spain Tuberculosis and brucellosis are infectious diseases prevalent that produce similar focal complications, which create problems of differential diagnosis. Rapid detection of both pathogens is necessary for the timely initiation of antimicrobial treatment. The traditional techniques for both diseases present limitations and delays in diagnosis. Furthermore, they can be very insensitive in some cases of extrapulmonary tuberculosis and focal forms of brucellosis. The aim of this study was to develop a multiplex real-time PCR (M RT-PCR) assay, using novel targets, to simultaneously


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Abstracts detect Brucella spp and Mycobacterium tuberculosis complex (MTC) and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and Omp2a genes was used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes was targeted for the detection of members of the MTC. The analytical sensitivity of assay was performed from pure cultures strains of Brucella abortus B-19 and Mycobacterium tuberculosis H37Rv, either independently or jointly. The specificity was confirmed using different strains of Brucella, MTC and non-tuberculosis mycobacteria. A total of nine reactions M RT-PCR based on SYBR Green I was evaluate by using the different primers combinations. A test was defined as positive only when the gene combinations were capable of co-amplifies both pathogens in a single reaction tube and showed melting temperature distinguishable for each microorganism. The best preliminary results correspond with M-RT-PCR using bcsp31 and senX3-regX3 genes for the identification of Brucella and MTC, respectively. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate and sensitive to the detection of human tuberculosis and brucellosis.

YSF.106 Free-R-methionine sulfoxide reductases: kinetic characterization of the three steps and of aminoacids involved in catalysis and substrate specificity M. Libiad, G. Branlant and S. Boschi-Muller AREMS UMR 7214 CNRS-UHP, Nancy Universite´, Vandoeuvrele`s-Nancy, France Methionine sulfoxide reductases (Msr) catalyze the reduction of methionine sulfoxides (Met-O) into Met, in the presence of reducer. This family of enzymes consists of three structurally unrelated classes, of which one, recently discovered and called fRMsr, selectively reduces the free form of Met-R-O and belongs to the GAF-fold family. GAF proteins are usually involved in cellular signaling. fRMsrs represent the first GAF domain to show enzymatic activity. Previous studies of our group support for the fRMsrs a three-step mechanism with at least formation of a sulfenic acid on the catalytic Cys118 in the first step and then of a disulfide bond, which is reduced by thioredoxin in the last step. Inspection of the 3D structure of the N. meningitidis fRMsr in complex with its substrate shows the presence of: (i) an oxyanion hole, (ii) an hydrophobic pocket and (iii) a network of hydrogen bonds between acidic amino acids and including a water molecule. In the present study using kinetic approaches, including stopped-flow and quenched-flow under single turn over conditions, and adapted mutants, the molecular factors involved in catalysis and substrate specificity are characterized.

YSF.107 Inhibition of fibrin clot formation with sulfated cellulose from Po´pulus tre´mula wood E. Savchik, N. Drozd, S. Kuznetsova, V. Makarov, V. Levdanskiy and B. Kuznetsov FEBS Society in Russia Since 2010 At present time is actual the search of compounds with anticoagulant (AA) and antithrombotic activities that are alternative to heparin. Sulfated polysaccharides (SP) of plant or animal origin are very promising and their AA depends on structure parameters. Six samples of sulfated cellulose (SC) from Po´pulus tre´mula wood with different molecular weight (Mr, 12–19 kDa), polymerization

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YSF – Young Scientist Forum degree (PD, 57–87), sulfation degree (SD, 0.54–0.61) and sulfur content (S, 8.0–8.7%) were investigated in vitro with standard coagulation assays: activated partial thromboplastin time (aPTT), Hep-test, prothrombin time (PT), thrombin clotting time (TCT), which were done in citrated plasma. International unfractionated heparin (UFH) NIBSC standard with antithrombin activity (aIIa) 203 U/mg and anti-factor Xa (aXa) activity 220 U/mg was used as comparison sample. In tests aPTT, Hep-test, PT, TCT the clotting time was prolonged by increasing concentrations of studied SC. The most active compounds in aPTT showed aIIa = 45.6 ± 1.0– 53.8 ± 6.8 U/mg, it was 3.77–4.45 times less than UFH had. In Hep-test SP displayed aXa = 8.6 ± 1.8–10.5 ± 2.0 U/mg, which in 20.95–25.58 times was less than UFH had. In PT for certain effect we needed the most active SC with concentration 4.46–5.15 times higher than UFH’s and in TCT – 4.64–6.54 times. In every assay a compound with Mr = 14 kDa, SD = 0.55, PD = 64 and S = 8.1% was the least effective: in aPTT and Hep-test the one was 3.5–5 times less active and it was taken 10 times more than others in PT and TCT. Reliable relationship aIIa or aXa with parameters of SC (Mr, SD, PD, and S) wasn’t detected. In range of Mr 12.7–18.9 kDa SC from Po´pulus tre´mula wood didn’t significantly differ in their AA. Less AA the sample with Mr = 14 kDa and S = 8.1% can be explained by low sulfur content as a SP with lower Mr (12.7 kDa) and higher S (8.5%) has aIIa 4.3 times more. By comparison between two samples with same S (8.3%) the SC with more Mr is more active as anticoagulant.

YSF.108 Structure-function studies of m1A1408 aminoglycoside-resistance ribosomal RNA methyltransferases M. Savic1,2, R. Macmaster1, N. Zelinskaya1, S. Subramanian1, P. Desai1, B. Vasiljevic2 and G. L. Conn1 1 Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA. 2Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe, Belgrade, Serbia Global spread of aminoglycoside resistance methyltransferases (MTs) among pathogens threatens usefulness of these antibiotics in clinical practice. Modifications of 16S ribosomal RNA (rRNA) to N1-methyl A1408 (m1A1408) imparts high-level resistance to a broad spectrum of aminoglycosides, including examples of each structural class. Guided by X-ray crystal structure, we have experimentally defined the critical residues for S-adenosyl-methionine (SAM) binding and an extended protein surface on the A1408 MT KamB that probably interacts with the 30S ribosomal subunit. Despite low protein sequence similarity between enzymes discovered in antibiotic producing and pathogenic bacteria, the structures of three enzymes from diverse species, KamB, Kmr and NpmA, are essentially identical. Small differences occur in two extended structures, unique to this family of enzymes that link the final three b-strands of the core SAM-binding domain and probably control their target site recognition. The loop connecting b-strands 5 and 6 appears to have dynamic extended structure that we previously proposed might mediate ‘‘flipping’’ of A1408 from 16S rRNA helix 44. This loop is positioned adjacent to both the bound SAM and interacts with a second unique helical loop motif between b-strands 6 and 7 that contains essential conserved amino acids for methyltransferase activity (e.g. W193 in KamB). The structure of Kmr also suggests that the b6/7 loop that forms parts of the SAM-binding pocket may also be dynamic in structure. An unexpected peculiarity of these enzymes is their substantially higher binding affinity for the


YSF – Young Scientist Forum methylation reaction by-product, S-adenosyl-homocysteine (SAH), over co-enzyme SAM. These findings suggest that concerted conformational changes in 16S rRNA and the protein provide a unique mechanism to control target selection specificity and activation of methyltransferase activity by promoting SAM binding.

YSF.109 Diagnostic usefulness of SCC, CA 125 and CEA in tumor discovery N. Serdarevic1,2 1 Institute for Clinical Chemistry and Biochemistry, University of Sarajevo Clinics Center, Sarajevo, Bosnia and Herzegovina, 2 Faculty of health sciences, University of Sarajevo, Sarajevo, Bosnia and Herzegovina Background: The study was to determined concentrations of total cholosterol, HDL cholesterol, apolipoprotein B-100 (apo B), apolipoprotein A (apoA) and Lpa at patients’ serum with vascular dementia. Methods: The study included 100 subjects, 50 patients diagnosed with vascular dementia developed as a consequence of ischemic brain stroke, i.e. of many small ischemic focus of various age and 50 healthy subjects. Lipids were determined using DIMENSION LxR automatic analyser of DADE BEHRING. The apolipoproteins were determined using SIEMENS automatic analyser (imunonefelometry). Results: Our results show that the concentration of HDL cholesterol and apo A were significantly lower in the group with and vascular dementia than in the control group. Average concentrations of cholesterol, apo B and Lpa were significantly higher in the group with vascular dementia than in the control group. In the group with vascular dementia, the concentration of apo B was higher than 1.33 g/l in about 60% (mean concentration 1.35 g/l) of patients then in control group whereas the concentration of Lpa was higher than the upper reference value in about 20% of patients (mean concentration 0.37 g/l). The mean concentration of cholesterol was significantly higher in the group with vascular dementia. Conclusions: Increasing concentrations of apo B and Lpa in the serum of patients with vascular dementia affects the further development of atherosclerosis and the development of new stroke.

YSF.110 Comparison of expression vectors for transient expression of recombinant proteins in plants K. H. Shah and H. Bohlmann Institute of Plant Protection, University of Natural Resources and Life Sciences (BOKU) Vienna, Austria Production of recombinant proteins in plants is getting more and more importance for practical applications. Transient expression vectors are efficient tools for this purpose. To date, a large number of such vectors have been constructed. Each of them is reported to be highly efficient, robust and cost effective which makes it difficult to choose the best vector. Therefore, we have undertaken a comparative analysis of a variety of available expression vectors, including pJLTRBO, pPZP3425, pEAQ-HTGFP, and pBY030-2R. In addition, we transferred the TMV expression cassette from pJLTRBO, into the pPZP vector backbone to make pPZP5000. We compared the expression of GFP and GUS in Nicotiana benthamiana by Agrobacterium-mediated transformation. In our hands pJLTRBO and pPZP5000 had a comparable expression

Abstracts level without RNAi inhibitor. The other vectors needed co-infiltration of an RNAi inhibitor expression construct to give good expression levels. The non viral vector pPZP3425 gave also satisfactory results when co-infiltrated with RNA silencing inhibitor P19.

YSF.111 LXRs reverse TNFa-dependent downregulation of ABCA1 gene expression in HepG2 cells V. S. Shavva, D. A. Mogilenko, E. B. Dizhe, S. V. Orlov and A. P. Perevozchikov Department of Embryology, St. Petersburg State University, St. Petersburg, Russia,Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, St. Petersburg, Russia ATP-dependent cassette transporter A1 (ABCA1) participates in creation of high density lipoproteins (HDLs), which exhibit antiatherosclerotic effects. As nascent HDLs are mainly formed in liver, we have studied expression of ABCA1 gene in human hepatoma cell line HepG2. As LXRs and PPARs are well-known regulators of lipid, we have examined the influences of these nuclear receptors on TNFa-dependent downregulation on ABCA1 gene expression in HepG2 cells. As TNFa activates MAP-kinase and NF-kB signaling cascades, we treated HepG2 cells with specific inhibitors of these pathways. Inhibition of NF-kB and p38 activity had no effect on TNFadependent ABCA1 gene suppression, while inhibition on MEK and JNK pathways resulted in increase in ABCA1 expression. Inhibition of JNK pathway abrogated TNFa-driven decrease of ABCA1 gene activity. LXRs abolishes TNFa-dependent decrease of ABCA1 gene expression in HepG2 cells. Treatment of HepG2 cells with LXRs or PPARc agonists lead to elevation of ABCA1 gene expression. Administration of PPARa agonist to HepG2 cells abolished downregulation of ABCA1 expression by TNFa, while LXRs and PPARc agonists triggered increase in ABCA1 gene transcription in HepG2 cells treated with TNFa. These effects might be due to increase in LXRa, LXRb and PPARa genes expression in cells, treated by PPARa and PPARc agonists and formation of positive regulation loop in cells, treated by LXRs agonist, which results in increase of LXRa gene expression. Because expression of PPARc gene in HepG2 cells, administered to LXRs agonist, is downregulated while expression of PPARa does not change, we believe that increase in ABCA1 expression in such tells is due to direct effects of LXRs on its transcription, while PPARs might exhibit their effects through upregulation of LXRs genes expression. We have shown that LXRs activation abrogates proimflammatory effects of TNFa on HepG2 cells, abolishing TNFa-dependent downregulation of ABCA1 gene expression.

YSF.112 GCP-2 expression by murine melanoma cells favours the incidence of metastases L. E. Sima1, V. Negoita2, I. Gruia2, G. Opdenakker3 and S. M. Petrescu1 1 Institute of Biochemistry, Romanian Academy, Bucharest, Romania2Al. Trestioreanu Institute of Oncology, Bucharest, Romania,3Rega Institute for Medical Research, Laboratory of Immunobiology, Leuven, Belgium Angiogenesis is a process that favours tumour growth by the formation of new blood vessels which bring nutrients to cancer cells and are also used as waste disposal pathway. Granulocyte chemotactic protein-2 (GCP-2) is an ELR + CXC chemokine


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Abstracts described as neutrophil chemoattractant and pro-angiogenic factor. It is secreted by many types of carcinoma cells from osteosarcoma, lung, prostate, breast and gastrointestinal tumours. However, the function of GCP-2 in tumour development is still not well understood. Previous investigations have shown that overexpression of murine GCP-2/CXCL5 (muGCP-2) in human melanoma cells stimulated tumour growth in vivo early in its development. In the present study we analyzed the effects of stable muGCP-2 expression over B16F1 murine melanoma cells invasivity in vitro and metastatic potential in vivo. Three clones stably expressing GCP-2 but differing in the amount of secreted chemokine have been investigated. Interestingly, the most pigmented clone displayed the highest GCP-2 transcription level. GCP-2 promoted the formation of peritoneal metastases in mice injected s.c. with melanoma cells independent of their pigmentation level. However, primary tumors’ volume was directly proportional to cell proliferation potential but did not correlate with the amount of secreted chemokine. Cells isolated from hyperpigmented metastases showed increased resistance to hypoxia, since incubation in 0.5% pO2 did not disturb either their proliferation rate or their viability. Their capacity to secrete GCP-2 was maintained during the metastasis process. Cells expressing higher levels of GCP-2 secreted increased amounts of active gelatinases and gave rise to a higher number of colonies in soft agar. Our results suggest that GCP-2 favours melanoma metastases development by increasing cells aggressive phenotype.

YSF.113 Functional analysis of copper transporter ATP7B in glial cell line U87 N. Sirocova, E. Scvortova1, N. Mocanu1, V. Sauer2, R. Siaj2, T. Todorov2, A. Zibert2, V. Sacara1 and H. H.-J. Schmidt2 1 National Center of Reproductive Health and Medical Genetics, Chisinau, Moldova, 2Klinische und Experimentelle Transplantationshepatologie, Universita¨tsklinikum Mu¨nster, Mu¨nster, Germany The Wilson disease protein (ATP7B) is a copper-transporting member of the P-type ATPase superfamily, which plays a central role in the regulation of copper homeostasis in the liver by delivering copper to the secretory pathway and mediating export of excess copper into the bile. The function of ATP7B in other organs is much less clear. Yet little is known about the specific role of ATP7B in the brain that can be assumed by frequent neurological manifestations in Wilson’s disease patients. Glial cells are characterized by low level of ATP7B expression. Using retroviral transduction we have generated 12 U87 cell lines expressing different ATP7B mutations. Mutations are located in the copper binding domain (n = 2), TM (n = 4), TGE loop (n = 1), ATP binding loop (n = 5). Functional characterization was done to determine whether transgenic expression of ATP7B mutants corresponds to clinical neurologic manifestations. MTT assay for 12 U87 ATP7B mutants including positive and negative controls was performed after 48 hour incubation with 0.06–1 mM copper. All samples were analyzed in triplicate. The transgenic ATP7B level was assessed by RTqPCR. At the functional level ATP7B mutants fell into two groups: mild or severe. All cells expressing mutations associated with early or late neurological manifestations showed comparable resistance to copper. Six from seven early onset mutations with various clinical manifestations located in the copper binding domain (n = 1), TM (n = 3) and ATP binding loop (n = 3) revealed low copper resistance. These findings show that functional analysis of glial cells may be helpful in prediction of the disease course and require further study.

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YSF – Young Scientist Forum YSF.114 Screening of protein hydrolysates for their potential to activate the CCK1 receptor D. Staljanssens, W. De Vos, J. Van Camp and G. Smagghe Faculty of Bioscience Engineering, Ghent University Cholecystokinin (CCK) is a gastrointestinal hormone and neuropeptide, which is involved in many physiological processes like satiety induction, gallbladder contraction and motility of the gastrointestinal tract and it exerts its functions by binding to the cholecystokinin receptor (CCKR). Molecules that mimic the effect of CCK can thereby influence CCKR-mediated processes. We have established a cell-based bioassay with CHO cells overexpressing the rat CCK1R, to screen for such molecules. This assay is based on the measurement of intracellular Ca2+ increases which arise from agonistic activation of this receptor. The Ca2+ increase was quantified using a fluorescence plate reader and subsequently validated with a confocal microscope to distinguish specific from nonspecific aspects and intercellular variability. This way, different enzymatic hydrolysates from food protein were screened for their potential to activate the CCK1R. Gel filtration was applied to the active hydrolysates in order to obtain fractions with a higher activity. Bioactive peptides which show CCK1R activity might be used as an ingredient for new nutraceuticals and functional foods that induce satiety as an aid in the battle against obesity or that can be used in the treatment of other CCKR involved diseases.

YSF.115 (S10.2.6) Evaluation of metabolic status in type 2 diabetes mellitus patients by proton nuclear magnetic resonance spectroscopy method (1H-NMR) L. I. Stefan1, A. Nicolescu2, S. Popa3, M. Mota4, E. Kovacs5 and C. Deleanu6 1 Department of Biochemistry, Craiova County Clinical Emergency Hospital, Craiova, Romania, 2Petru Poni Institute of Macromolecular Chemistry, Group of Biospectroscopy, Iasi, Romania, 3Department of Diabetes and Metabolic Diseases, Craiova County Clinical Emergency Hospital, Craiova, Romania, 4 Department of Diabetes and Metabolic Diseases, University of Medicine and Pharmacy, Craiova, Romania, 5Department of Medical Biophysics and Cellular Biotechnology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, 6 C.D.Nenitescu Institute of Organic Chemistry, Bucharest, Romania In spite of the great number of published papers on nuclear magnetic resonance (NMR) urine analysis, and the existence of several groups around the world active in the field, there are only a few published results on metabolites determined in type 2 diabetes mellitus (T2DM). In the first study we compared the value of the concentrations of different metabolites between normal and T2DM subjects trying to identify possible metabolic disorders of type 2 diabetes mellitus. In the second section the patients with T2DM were evaluated according to gender, body mass index and duration of diabetes mellitus and the NMR profile of metabolites concentrations established. Serial urine samples of 104 healthy subjects and 121 T2DM patients were examined by proton nuclear magnetic resonance spectroscopy (1H-NMR). The 1H-NMR spectra were recorded on a Bruker Avance DRX 400 MHz spectrometer operating at 9.4 Tesla. To 0.9 ml urine, 0.1 ml of stock solution of 5 mM sodium 3-(trimethylsilyl)-[2, 2,


YSF – Young Scientist Forum 3, 3-d4]-1-propionate (TSP) in 10% D2O has been added. Statistical analysis was performed with CBStat 5.1. The results are evaluated in mmol/mol of creatinine. A significant difference between the excretion of 3-hydroxyisovaleric acid, hippurate, creatine, valine, alanine, c-aminobutyrate, glycine, trimethylamine-N-oxide and citrate between healthy subjects and T2DM patients was found. There is a gender-related difference in the excretions of lactate and c-aminobutyrate between males and females in T2DM patients. We obtained that the concentrations of valine, lactate, pyruvate and trimethylamine in diabetics increased with the increase of body mass index. Major differences in the dynamic of metabolites excretion are signalized for alanine, citrate and dimethylamine in newly diagnosed T2DM patients. 1H-NMR could provide a tool to explore the presence of metabolites and their concentrations in the urine samples and to obtain information about the diseases status in T2DM patients.

YSF.116 Molecular interactions between microsomal cytochromes CYP17A1 and CYP21A2 involved in adrenal steroid biosynthesis T. A. Sushko, A. A. Gilep and S. A. Usanov Institute of Bioorganic Chemistry, National Academy of sciences of Belarus Protein-protein interactions play a significant role in regulation of functional activity of P450s, but few studies examined P450s catalyzing steroid synthesis. In the present study, the molecular interactions between microsomal cytochromes CYP17A1 and CYP21A2 associated with synthesis of corticosteroid hormones in adrenal cortex were analyzed in mixed reconstituted system. We investigated dependence of the CYP17A1-CYP21A2 interaction on NADPH-cytochrome P450 reductase (CPR) concentration. It is shown that presence of CYP17A1 in the reconstituted system containing CYP21A2 inhibit enzymatic activity of CYP21A2 at subsaturating reductase concentration, enzymatic activity of CYP21A2 at higher reductase concentration reaches the level of CYP21A2 activity in the simple reconstituted system. Analogously, presence of CYP21A2 in the reconstituted system containing CYP17A1 strongly inhibit enzymatic activity of CYP17A1 at subsaturating reductase concentration, but enzymatic activity of CYP17A1 at higher reductase concentration couldn’t reach the level of CYP17A1 activity in the simple reconstituted system. Furthermore the interaction between CYP17A1 and CYP21A2 was studied under the physiological concentration of proteins. The effect of the ionic strength on CYP17A1CYP21A2 interaction was studied. It is demonstrated that electrostatic contacts play role in this protein-protein interaction. Summarizing, we have shown for the first time that molecular interactions between CYP17A1 and CYP21A2 can modulate their catalytic rates.

YSF.117 Acute and chronic CNS effects of mildronate, an inhibitor of L-carnitine biosynthesis B. Svalbe1,2, L. Zvejniece1, E. Liepinsh1 and M. Dambrova1 1 Latvian Institute of Organic Synthesis, Riga, Latvia, 2University of Latvia, Riga, Latvia Mildronate (3-(2,2,2-trimethylhydrazinium) propionate; meldonium) is used in clinical practice as an anti-ischemic drug for cardiovascular and neurological applications including the treatment of patients with ischemic disorder of cerebral circulation. This study was undertaken to investigate the effects of

Abstracts mildronate in rats following middle cerebral artery occlusion (MCAO). The experimental models of anticonvulsant activity testing were used to investigate the possible mechanisms of acute activity of mildronate. Mildronate treatment (100, 200 mg/kg, intraperitoneal (i.p.) administration for 14 days after 90 minutes MCAO) was found to accelerate the functional neurological recovery of motor and proprioception deficits in limbplacing and beam-walking tests. Analysis by UPLC/MS/MS of rat cerebellar tissue extracts revealed that mildronate significantly decreased the concentration of L-carnitine. Cerebellar concentrations of mildronate increased in a dose-dependent manner. The results from sham-operated and saline-treated MCAO groups showed that surgery alone did not affect L-carnitine concentration in rat cerebellum. Mildronate treatment did not affect the infarct size. Mildronate (50–200 mg/kg, acute i.p. administration) exerted a dose-dependent anticonvulsant activity in a chemoconvulsant pentylenetetrazole-induced clonic and tonic seizure test but did not change the effects of a convulsion-inducing dose of (+)-bicuculline, an antagonist of c-aminobutyric acid receptors. The anticonvulsant activity of mildronate was completely blocked after pre-treatment with a2adrenergic receptor antagonist yohimbine (2 mg/kg) and nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (10 mg/kg). These results demonstrate that the chronic treatment of mildronate decreases L-carnitine concentration in brain tissues and improves the functional outcome in MCAO rats. After acute administration of mildronate the anticonvulsant effect involves the a2-adrenergic and NO-dependent mechanisms.

YSF.118 Misfolded protein oligomers formed by a model protein mimic the effect of a-beta oligomers on synaptic plasticity involved in Alzheimer’s disease F. Tatini, A. M. Pugliese2, C. Traini2, S. Niccoli1, G. Maraula2, T. Ed Dami2, B. Mannini1, F. Pedata2, F. Casamenti2 and F. Chiti1 1 Biochemistry Department, University of Florence, Viale Morgagni, Florence, 2Pharmacology Department, University of Florence, Viale Morgagni, Florence The oligomeric assemblies forming during the conversion of proteins from their native state into fibrillar aggregates are thought to be the primary pathogenic species in many protein deposition diseases. Oligomers by the amyloid-beta (A-beta) peptide cause neuronal dysfunction in Alzheimer’s disease (AD), by acting through specific mechanisms, i.e. by binding to the neurons at the level of the synapses, inhibiting Long Term Potentiation (LTP), and impairing cognitive functions. We have previously shown that protein oligomers formed by a protein that has no link to human disease, namely HypF-N, is able to cause generic cell dysfunction. Another type of HypF-N oligomers formed under different conditions were found to be harmless. We called these oligomers type A and B, respectively. We investigated whether the HypF-N oligomers have a specific pattern of neuronal damage similar to that of A-beta oligomers and whether the two HypF-N oligomers have a different effect. To this purpose, we tested if type A and B oligomers (i) co-localise with post-synaptic densities, (ii) inhibit LTP and (iii) impair spatial learning. Our experiments show that type A oligomers mimic the effect of amyloid-beta oligomers in all tests, i.e. they co-localise with postsynaptic densities in rat hippocampal cultured neurons, inhibit LTP in the CA1 region of rat hippocampal slices and impair spatial learning when injected into rat’s brains. Type B oligomers


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Abstracts were not found to have any of such effects, similarly to the native protein. These results show that toxic oligomers formed by a model protein can mimic the toxicity of A-beta aggregates. These findings raise the importance of using HypF-N oligomers as a valid tool to investigate the pathogenesis of AD, but also support the concept that formation of multiple types of protein aggregates resulting from a failure of protein homeostasis can result into a number of species with similar effects and thus cooperating in the pathogenesis of disea

YSF.119 Regulation of the canonical Wnt pathway by endocytic proteins A. Torun, S. Rashid, E. Szymanska, I. Pilecka and M. Miaczynska Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland The canonical Wnt pathway has a great impact on many biological processes such as development, stem cell maintenance and carcinogenesis. Studies concerning the role of endocytic proteins in this pathway are rather conflicting, often showing opposite effects. These discrepancies may arise from the fact that endocytic proteins can regulate cell signalling in many different ways. First of all, endocytosis is a process by which activated receptors are removed from the cell surface. Secondly, signal transduction may continue after internalization of receptors and different accessibility of downstream signalling molecules on endosomes may change the overall signalling output. Finally, some endocytic proteins are present also in the cell nucleus where they may affect gene transcription. Endocytic adaptor proteins APPL1 and APPL2 constitute an example of such a dual function proteins. Under certain conditions APPL proteins translocate to the cell nucleus and can interact with a transcriptional regulator Reptin. Reptin in complex with a transcriptional co-activator of the Wnt pathway, b-catenin, acts as a repressor because it concomitantly recruits histone deacetylases (HDACs). We revealed that APPL proteins modulate the composition of the b-catenin-Reptin-HDAC complex and thereby relieve Reptin-mediated repression of the b-catenin/ TCF target genes. As the alternative roles of endocytic proteins in the canonical Wnt pathway have never been studied in a systematic way we are extending our studies to other endocytic proteins besides APPLs. We have set up a luciferase-based RNAi screening to carefully study involvement of 82 selected proteins in the b-catenin/TCF-dependent transcription. We plan to test if and how endocytic and non-endocytic functions of novel regulators identified in our screen interplay in the control of the canonical Wnt signalling.

YSF.120 Structural insights on a new tetracycline resistance mechanism relying on the TetX monooxygenase G. Volkers, G. J. Palm, M. S. Weiss, G. D. Wright and W. Hinrichs Institute for Biochemistry, Molecular Structural Biology, ErnstMoritz-Arndt-Universita¨t Greifswald, Greifswald, Germany The flavin-dependent monooxygenase TetX2 from anaerobic Bacteroides thetaiotaomicron confers resistance against tetracyclines on aerobically grown Escherichia coli [1]. The enzyme modifies several tetracycline antibiotics including the recently

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YSF – Young Scientist Forum approved 3rd generation antibiotic tigecycline under regioselective hydroxylation of the substrate which leads to non-enzymatical degradation associated with weaker antibiotic properties [2]. In contrast to efflux or ribosomal protection mechanisms, this resistance mechanism is only partly understood. TetX2 has also been found in aerobic Sphingobacterium sp. which may be the ancestral source of the tetX-genes. The crystal structure was solved in a 3-WL MAD experiment with a SeMet-containing crystal in space group P21. The native protein crystallized in P1 and data were collected to a resolution of 2.1 A˚. TetX2 shares highly conserved homologous domains with other structurally known FAD-binding monooxygenases like PhzS hydroxylase, despite low sequence identity and different substrates. The substrate binding domain contains a seven stranded b-sheet. Two strands connect the FAD-binding domain with the substrate binding domain additional to a helix composed of eight turns. To gain insights into the molecular basis of drug degradation we performed crystal structure analyses of TetX in complex with its substrates. The structure of the TetX:7-iodotetracycline and TetX:7-chlorotetracycline complexes was solved to a resolution of 2.3 A˚ and reveals important details on binding and recognition as well as degradation of tetracycline antibiotics. In this rare case binding of tetracyclines to an active site occurs independently from magnesium. References: 1. Yang W., Moore I.F., Koteva K.P., Bareich D.C., Hughes D.W., Wright G.D., JBC 2004; 279 (50): 52346. 2. Moore I.F., Hughes D.W., Wright G.D., Biochemistry 2005; 44: 11829.

YSF.121 Hyperphosphorylation of intrinsically disordered Starmaker protein increases its biomineralization activity M. Wojtas and P. Dobryszycki Department of Biochemistry, Wrocaw University of Technology, Wrocaw, Poland Formation of biominerals, like bone, teeth or shell is strictly regulated by proteins, especially by highly acidic phosphoproteins. Otoliths existing in fish inner ear are mineral deposits, responsible for gravity sensing and the perception of sound. Otoliths are composed of calcium carbonate associated with small fraction of organic molecules which act as a template for crystals deposition, by promoting the growth of crystal in desired directions and controlled inhibition of their growth. Starmaker (Stm) controls size, shape and polymorph of calcium carbonate of otoliths formation in Danio rerio. Stm, like other proteins involved in biomineralization is highly acidic (Asp + Glu 36%) rich in Ser residues (19.5%). Unusual amino acid composition results in very extended, rod-shaped conformation, hence Stm is one of intrinsically disordered proteins (IDPs) which exhibit extraordinary structural flexibility and lack of well-defined tertiary structure. However, the Stm conformation undergoes compaction induced by calcium ions, which are putative ligands of Stm. We have designed in vitro biomineralization assay combined with scanning electron microscopy which has indicated that Stm modulates growth of calcium carbonate crystals. We have shown that CK2 causes hyperphosphorylation of Stm. It is clearly demonstrated that Stm exhibits high biomineralization activity and phosphorylation significantly increases this ability. We also have used CD spectroscopy to show the effect of phosphorylation by CK2 and/ or calcium ions on the Stm secondary structure. Acknowledgements: This work was supported by Polish Ministry of Science and Higher Education and by the Wrocaw University of Technology.


YSF – Young Scientist Forum YSF.122 Widespread regulation by cis-antisense transcripts in the virulence genome of Pseudomonas aeruginosa O. Wurtzel1, D. Yoder-Himes2, S. Edelheit-Flohr1, S. Lory2 and R. Sorek1 1 Weizmann Institute of Science, Rehovot, Israel, 2Harvard Medical School, Boston, MA, USA Pseudomonas aeruginosa is an opportunistic pathogen that thrives in diverse environments including soil, plants, and animals. Its complex genome displays a mosaic of core components and nonconserved genomic islands accounting for its infectious and metabolic diversity. Although the transcriptome of P. aeruginosa has been extensively analyzed using DNA microarrays, little is known about the expression and function of the non-coding portions of its genome. We used whole transcriptome sequencing, combined with large-scale transcription start site mapping, to study the non-coding transcriptome of P. aeruginosa grown at 37C (human body temperature) and 28C. Our data show that the P. aeruginosa transcriptome is populated by over 400 cis-antisense RNA transcripts, many of which overlap pathogenicity and motility-related genes and are therefore likely to function in virulence regulation. One of these cis-antisense transcripts was found to overlap the alginate biosynthesis operon, a key pathogenicity gene responsible for the mucoid phenotype of strains isolated during the late-stage cystic fibrosis disease. When we knocked out antisense transcription in this gene we observed altered mucoid behavior, clearly indicating the involvement of cis-antisense in alginate regulation. Our data also reveals a widespread, distinct class of cis-antisense RNAs overlapping 5¢ ends of protein coding genes, which may regulate translation by sequestering the Shine-Dalgarno sequence. These results imply that cis-antisense transcription is an important regulation strategy of the P. aeruginosa in its natural environment and during infection.

YSF.123 New date about the existence of hypothalamus-bone marrow neurohumoral axis. Participation of NPV in regulation of carbohydrate phosphorus metabolism in bone marrow and spleen L. A. Yeranosyan, L. P. Ter-Tatevosyan and A. A. Galoyan 2010 Taking into account a huge body of experimental evidence concerning the PRP stimulatory action on carbohydrate, protein, and lipid metabolism, we decided to study the role of hypothalamic neurosecretion in the regulation of certain enzymes of carbohydrate-phosphorus metabolism. The purpose of the present study was to elucidate the existence of hypothalamus-bone marrow neurohumoral axis by assessment of the enzymatic activities of acid and alkaline phosphatases and glycogenphospholylase. Pharmacological desymphatized rats were exposed to irritation of NPV nuclei of hypothalamus, and further enzymatic activities of acid and alkaline phosphatases and glycogenphospholylase were measured in hip bone marrow and spleen tissues. The catecholamine-specific neurotoxin 6-hydroxydopamine (6-OHDA) has been used to selectively destroy dopaminergic and noradrenergic neurons. The effects of 3 seconds electrical stimulation of PV nuclei were investigated in symphatectomized, adult, male rats. A sensitive and rapid spectrophotometric method is used for determination the enzyme activity. We found that suppression of the sympathetic nervous system does not affect glycogen phosphorylase and substantially

Abstracts increases the activity of acid phosphatase (by 92%) in the bone marrow. In the spleen, these enzymes are more resistant to sympathectomy. Electrical stimulation of the PVN produced 50% increase of in activity of acid phosphatase in bone marrow. Not having activity in intact and sympathectomized rats, glycogen phosphorylase raised as much as possible in bone marrow reaching 1651 Unit. Data obtained indicate that electrical stimulation of paraventricular (PV) nuclei in simpathectomized rats reduce the effect of neurotoxin 6-OHDA, which was observed in changes of glycogen phosphorylase activity. From experimental data we come to know, that hypothalamic neurosecretion take a great part in regulation of carbohydrate metabolism in hematopoietic organs.

YSF.124 Homogeneous immunoassay using gold nanoparticles for diagnostic of systemic lupus erythematosus H. Yeritsyan and V. Gasparyan H. Buniatian Institute of Biochemistry of NAS, RA The sensitivity of biomarker compounds detection method in patient fluid samples has a critical importance in early diagnosis, treatment and management of various diseases. For solving of this problem different assay approaches have been developed and the system applying colloidal gold nanoparticles is one of those. The unique optical properties of colloidal gold nanoparticles make it possible to detect minor amounts of tested molecules/disease markers. Application of gold nanoparticles in diagnosis of Systemic Lupus Erythematosus is the subject of this investigation. We have applied ds-DNA-immobilized gold nanoparticles for detection of anti-DNA antibodies. These antibodies known also as antinuclear antibodies are appeared during some autoimmune diseases especially Systemic Lupus Erythematosus and therefore this approach can be useful for very sensitive and specific detection of this pathology. In the presence of appropriate antibodies, DNA covered gold nanoparticles agglutinate following with appropriate change in optical spectra. Rate of these changes correlates with amount of these antibodies. In this work the different approaches for immobilization and stabilization of gold sols were applied. Effects of various factors such as pH, ionic strength and amount of immobilized DNA, nature and amount of blocking agents on reaction were considered. Sensitivity and specificity of the assay was estimated and possible interfering substances and cross reactions with other diseases were considered. The system could be considered as a highly sensitive for detection of specific anti-DNA antibodies and therefore could be effectively used in diagnostics Systemic Lupus Erythematosus.

YSF.125 Structural, enzymatic and allergologic characterization of pollen allergen Phl p 4 D. Zafred1, A. Nandy2, M. Wald2 and W. Keller1 1 Institute of Molecular Biosciences – Structural Biology, University of Graz, Graz, Austria, 2Allergopharma J. Ganzer KG, Reinbek, Germany Background: To lower the risk and the levels of side effects of the allergen extract administration, new hypoallergenic derivatives should be created that evoke the immune response but do not cause mast cell degranulation. In order to create such hypoallergens in a rational manner the knowledge of IgE epitopes on the allergen surface is essential. Phl p 4 is one of the major allergens from timothy grass (Phleum pratense) and thus important cause of the hay fever as well as


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Abstracts other diseases that evoke from pollen allergy. This highly crossreactive allergen is recognized by 70–88% of grass pollen allergic patients and thus important part of potential medicament. Methods: Recombinant Phl p 4 has been produced in a eukaryotic expression system. 3D structure was determined by X-ray crystallography using the structure of berberine bridge enzyme for molecular replacement. Enzymatic properties of the allergen have partly been characterized and epitope prediction using the program SPADE is in progress. Results: The structure has been solved to 1.9 A˚ showing the fold of the BBE-like oxidoreductase with bi-covalently attached FAD. Glucose is oxidized by the enzyme using DCPIP as the electron acceptor showing, that the protein is glucose dehydrogenase with unknown function as the natural electron acceptor is not known to date. Two known linear epitopes have been mapped and putative conformational epitopes are being predicted by SPADE. Conclusion: We represent the first structure of a group 4 pollen allergen. In addition, the allergen has been partly characterized in enzymatic and allergologic manner.

YSF.126 Three MAP kinases of HOG signaling pathway from extreme halophilic fungus Wallemia ichtyophaga T. Konte, M. Lenassi, M. Fettich and A. Plemenitas Institute of Biochemistry, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia Sensing differences in environmental osmolarity is of essential importance to organisms, especially if they inhabit environments

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YSF – Young Scientist Forum with either fluctuating or high osmolyte concentrations (solar salterns, salt lakes, salt deserts etc.). Cell must maintain normal water potential and turgor, therefore sensing and reacting is crucial. Signaling pathway sensing osmolarity in Saccharomyces cerevisiae is known as high-osmolarity glycerol (HOG) and has been studied extensively in the past decades. Recently we identified HOG components of extremely halotolerant fungus Hortaea werneckii to get some insight into molecular adaptations to such environments. Our research is now aimed at HOG components in extremely halophilic basidiomycetous fungus Wallemia ichthyophaga, which is the most halophilic fungus known to date. Compared to H. werneckii, which does not require salt to remain viable, W. ichthyophaga needs at least 9% NaCl (w/v) in the media and is metabolically active even at saturated NaCl concentration. To identify HOG kinases, W. ichthyophaga cells were grown to exponential phase, mRNA was isolated and cDNA was reverse transcribed. Coding sequences were obtained from cDNA using PCR technique. Amino acid sequences were deduced out of coding sequences. Alignments, motifs and 3D structures were constructed using ClustalW, ELM, SwissModel and PyMOL software. For functional complementation assay we cloned W. ichthyophaga kinase genes into S. cerevisiae deletion strains using low copy plasmid under constitutive TPI promoter. With immunoprecipitation and western blot analysis we confirmed the presence of kinases cloned to transformants and determined Hog1 activation and phosphorylation. By identifying HOG pathway MAP kinase module [WiSte11 (MAPKKK), WiPbs2 (MAPKK) and WiHog1 (MAPK)], we confirmed the existence of HOG signaling pathway as well as its putative role in osmosensing in W. ichthyophaga.
