Báo cáo khoa học: YSF – Young Scientist Forum pot

YSF – Young Scientist Forum YSF.1 CV-IIL lectin: another piece of the Chromobacterium violaceum puzzle L. Adamova ´ 1 , J. Adam 1 and M. Wimmerova ´ 1,2 1 National Centre for Biomolecular Research, Kotla ´ r ˇ ska ´ 2, Brno, Czech Republic, 2 Department of Biochemistry, Masaryk University, Kotla ´ r ˇ ska ´ 2, Brno, Czech Republic One ot the most representative (sub)tropical soil microbe, Chro- mobacterium 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 struc- turally 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-lec- tin 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 isother- mal 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. Castan ˜ o Departamento de Bioquı ´ mica, Instituto de Investigaciones Biome ´ dicas ‘‘Alberto Sols’’, Universidad Auto ´ noma de Madrid y Consejo Superior de Investigaciones Cientı ´ ficas (UAM-CSIC), Centro de Investigacio ´ n Biome ´ dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Idipaz, Facultad de Medicina UAM, Madrid, Spain Intracellular deposits of aggregated alpha-synuclein are a hall- mark 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, promot- ing 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 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-syn- uclein 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 neu- rotrophic factor for neurons in culture. In fact, oleic acid pro- motes axonal and dendritic growth and the expression of the axonal growth-associated protein 43 (GAP43) and of the micro- tubule-associated protein 2 (MAP2). Our last results to date show that albumin promotes the extension of GAP43-positive ax- ons 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-pro- teins (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 neuro- trophic effect of oleic acid. 446 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. Aureli 1 , N. Loberto 1 , R. Bassi 1 , M. Filocamo 2 , V. Chigorno 1 , A. Prinetti 1 and S. Sonnino 1 1 Departement of Medical Chemistry, Biochemistry and Biotecnology for the Medicine, University of Milano, 2 S.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 conserva- tion 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 mod- ulated, as b-hexosaminidases, b-galactosidases and arylsulphata- ses. 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 glio- blastoma and normal adult human brain. Serial Analysis of Gene Expression found Chitinase 3-like 1 (CHI3L1, HC gp-39, YKL- 40) 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 activa- tion of extracellular signal-regulated kinases ERK1/2 (MAPK)- and Akt (PI3K)- mediated signaling cascades. Western-blot anal- ysis of 293 cells and glioblastoma derived U373 cells after treat- ment 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 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, asso- ciated 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 phe- notype, 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 dif- ferent 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 inhibi- tor, exhibiting estrogen receptor binding activation, DNA topo- isomerase 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 differ- entiation in several tumor cell models, including leukemia cells. However, the regulation and signaling of genistein-induced leuke- mia 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 geni- stein-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 fur- ther 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: acetylcho- linesterase (AChE, E.C. 3.1.1.7) and butyrylcholinesterase (BChE, E.C. 3.1.1.8). BChE is distinguished from AChE by sub- strate preferences and differential sensitivity to certain inhibitors It is toxicologically and pharmacologically important in scaveng- YSF – Young Scientist Forum Abstracts FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 447 ing and detoxifying cocain, organophosphorus pesticides, carba- mate 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, aim- ing 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 spectrophoto- metrically at 25 oC in 50 mM MOPS buffer pH 8, using butyryl- thiocholine as substrate. NB caused complex, nonlinear inhibition of wild type BChE and A328F mutant. With A328F BChE mutant, intrinsic K’ value ( ” [I] 2 0.5 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; butyrylcholinester- ase 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 struc- tures plays exopolymeric matrix – the polymer in which cells are immersed. Studying of the biofilms formed by corrosion danger- ous 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 monocul- ture 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 cul- ture. 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 compli- cation 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 carbo- hydrate in the structure were examined. The carbohydrate part of the bioflms matrix visualized by ConA + FITC (Sigma), specific for glucose, for cells visualiza- tion – 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 lay- ers of cells localized in the bottom carbohydrate layers. The lower layer of carbohydrates partially contained cells. This distri- bution was characteristic of both mono and for associative cul- 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 community- acquired infections. Its global success might in part be explained by its genetic transformation, which consists of the internaliza- tion and the incorporation in its chromosome of exogenous DNA. Transformation is turned on in cells which are in a physi- ological 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 ope- rons. 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 regu- latory domain, linked to a variable effector domain. The major- ity of RRs (63%) contain DNA binding effector domains. Among them, 5% interact with DNA through the recently char- acterized 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 with- out 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. Abstracts YSF – Young Scientist Forum 448 FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies Oxidative stress was monitored in kidney and liver by measur- ing 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 kid- ney 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 abnormali- ties 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 pathogene- sis, progression and metastasis of prostate cancer (PCa). How- ever, 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 cel- lular 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 man- ner. The sensitivity of PC3 cells to EGFR inhibitors, determined as IC50 values and the inhibition of the cell growth, was mark- edly 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 expo- sure 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 block- age might be circumvented by the alternative signalling through HER3 in the resistant cells. In conclusion, our results indicate that the ability of androgen- independent 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. 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 Prote ´ ines, 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 migra- tion by inducing formation of cytoskeleton related protrusive structures. We have shown that SYN1 mediated cell adhesion occurs in heparan sulfate and chondroitin sulfate dependent man- ner 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 con- served heparin binding residues throughout LNs, as well as ‘‘can- didate’’ 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 com- parable to that of the wild type protein. Heparin affinity chroma- tography 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 inter- action 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 gam biae 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 inter- play 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 regu- lating 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 YSF – Young Scientist Forum Abstracts FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 449 insights into the mechanism sustaining catalysis. We performed an enzymatic characterization of PfCDPK4 using a non-radioac- tive 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 inhib- itors. Overall, our results represent a basis for the development of small molecules interfering either with the mosquito or the para- site 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 dystro- phin 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 dere- gulates 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 pro- teins. 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 repres- sion 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 dif- ferentiation. YSF.15 (S3.2.6) The signal peptides and the early mature domain cooperate for efficient secretion K. I. Chatzi 1,2 , G. Gouridis 1,2 , G. Orfanoudaki 1,2 , M. Koukaki 2 , I. Tsamardinos 3 , S. Karamanou 2 and A. Economou 1,2 1 Department of Biology, University of Crete, Crete, Greece, 2 Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology-Hellas, Crete, Greece, 3 Institute of Computer Science, Foundation of Research and Technology-Hellas, Crete, Greece 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 energy and activating the translocase. The activation of the tran- slocase machine and the trapping of the preprotein in the chan- nel, 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 (Alka- line phosphatase) gene and the in vivo and in vitro secretion effi- ciency 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 pep- tide 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. Ciaudo 1,2 , J. Toedling 3 , I. Okamoto 2 , N. Servant 3 , E. Barillot 3 , E. Heard 2 and O. Voinnet 1 1 Swiss Federal Institute of Technology (ETH-Z), Zurich, Switzerland, 2 Institut Curie, CNRS UMR3215, rue d’Ulm, Paris, France, 3 Institut 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 popula- tions 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, includ- ing genes. Some of these small RNAs have characteristics of cog- nate 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 R. Dakarviene ¨ , L. Degutyte ¨ -Fomins, R. ‡u ˆ kiene ¨ , V. Mildaþiene ¨ and Z. Naue ` iene ¨ Department of Biochemistry and Biotechnologies, Vytautas Magnus University, Centre of Environmental Research 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 Abstracts YSF – Young Scientist Forum 450 FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies clear correlation with Mt dysfunction and reactive oxygen species (ROS). We have focused our study on response of Mt from nor- mal 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 37–45°C temperature range. Internal ROS generation was determined fluo- rimetricaly with dihydrodichlorofluorescein diacetate (H2DCF- DA) and external – with Amplex Red. The results showed that at 37°C much less ROS are generated by female rat Mt, as com- pared 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 40°C (compared to 37°C) and 14% and 18% at 45°C, respectively; than 6–7 month rats ROS generation at 40°C was higher 14 ir 17% for male and female and 26% and 34% at 45°C respec- tively. At 45°C 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 45°C temperature – internal ROS content at 45°C was higher by 1.6 times comparing to that at 37°C. YSF.18 Functional analysis of the NF-jB signaling pathway protein TANK A. Dalmizrak 1 , F. Renner 2 and M. L. Schmitz 2 1 Department of Medical Biology, Faculty of Medicine, Ege University, Bornova, Izmir, Turkey, 2 Institute of Biochemistry, Medical Faculty, Justus-Liebig-University, Giessen, Germany NF-jBs are transcription factors that can be activated by cyto- kines, 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. Further- more, the protein which had the 10 amino acid changes and was also C-terminally truncated (DC95) again displayed a slight phos- phorylation. To understand the relationship between the TANK and SUMO1 proteins, four lysine amino acids in the TANK pro- tein sequence were mutated to arginine amino acids. After phos- phorylation 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-transla- tional modifications of the TANK protein will help us to further understand the role of this protein in the NF-jB signaling path- way and in the pathogenesis of diseases. 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 cat- alytic activity of those enzymes depends on the accessibility and affinity of the gaseous substrate to the catalytic centre, and, ulti- mately, its reactivity there. The structural determinants of these molecular events are most of the times inaccessible to experimen- tal approaches, specially the diffusion of dioxygen, where X-ray crystallography with xenon probes is probably the best experi- mental approach. In this case, biomolecular modelling and simu- lation 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 diffu- sion of dioxygen inside CotA laccase through molecular dynam- ics. 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 dioxy- gen 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/anti- inflammatory properties and apoA-I-containing subpopulations of HDL from family subjects with monogenic low HDL disorders G. Daniil 1 , A. A. P. Phedonos 1 , A. G. Holleboom 2 , M. M. Motazacker 2 , L. Argyri 1 , J. A. Kuivenhoven 2 and A. Chroni 1 1 Institute of Biology, National Center for Scientific Research ‘‘Demokritos’’, Athens, Greece, 2 Department of Experimental Vascular Medicine, Academic Medical Centre, Amsterdam, the Netherlands Abstract Background: Genetic factors regulate both high-density lipopro- tein (HDL) levels and functionality, thus affecting HDL antiath- erogenic properties. We characterized the HDL antioxidant/ anti-inflammatory properties and apoA-I-containing subpopula- tions in families with monogenic HDL disorders. Methods: Subjects with mutations in apolipoprotein A-I (apoA- I), ATP-binding cassette transporter A1 (ABCA1) or lecithin:cho- lesterol 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, mal- ondialdehyde (MDA), PAF and serum amyloid A (SAA) were measured. ApoA-I-containing HDL subpopulations were ana- lyzed by two-dimensional nondenaturing gel electrophoresis. Results: ApoA-I heterozygotes and subjects with partial or com- plete ABCA1 or LCAT deficiency had HDL with reduced antiox- YSF – Young Scientist Forum Abstracts FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 451 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 activ- ity was reduced in subjects with partial or complete ABCA1 defi- ciency 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 subpop- ulation. Some LCAT heterozygotes showed also reduction of the a2 subpopulation. Subjects with complete LCAT deficiency showed mostly the small a4 HDL subpopulation and with com- plete 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/anti- inflammatory properties of HDL. Furthermore, our study shows the effect of specific mutations on the apoA-I-containing HDL subpopulations profiles. YSF.21 Galectin-3 activates b1-integrins through CD98 G. Delgado, J. Fort and M. Palacı ´ n Institute for Research in Biomedicine-Barcelona, Universitat de Barcelona, Spain 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 a- amylases. CD98 constitutively associates with b1-integrins and plays an essential role in its activation: antibodies against CD98 block fibronectin-dependent integrin adhesion. We have demon- strated that galectin-3 activates AKT, thus leading to cell surviv- ing, 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 in- tegrin through CD98; using stem cell-derived fibroblasts knock out for CD98, recombinant proteins and fluorescent confocal microscopy. 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 nasopha- ryngeal 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 gov- erning 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 immunoprecipitation data evidenced about complex formation between LMP2A and ITSN1 in vivo in different cell types. SH3- domains 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 interac- tion of SH3-domains of ITSN1 with -PXXP- motives of LMP2A and ITAM-motives of LMP2A with SH2-domain of adaptor pro- tein Shb that bind simultaneously ITSN1 and LMP2A. Shb was shown to be phosphorylated and activated in this complex. Previ- ously, 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 ubiqu- itin-ligases AIP4 and c-Cbl target ITSN1 for degradation. More- over it was shown that AIP4 mediates ubiquitynation of ITSN1. Current findings provide new data about LMP2A-driving sig- nalosome assembly and functioning addressing question about coupling of its traffic and signaling activity. YSF.23 Microexon-based regulation of ITSN1 and Src SH3 domains specificity in brain M. Dergai, O. Dergai, I. Skrypkina, L. Tsyba, I. Zlatskiy and A. Rynditch Institute of Molecular Biology and Genetics, NASU, Kiev, Ukraine The SH3 domains function as protein-protein interaction mod- ules 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 dif- ferentiate. Further, structure modelling made it possible to uncover possible mechanism of regulation of SH3A domain bind- ing properties by microexon 20. The mechanism was suggested to rely on changes in position of charged amino acids within inter- action interface. Using mutational analysis we confirmed that this is realized to modulate ITSN1 (SH3A domain) binding to neu- ron-specific GTPase dynamin 1. Similar microexon-based mecha- nism 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.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 overex- pression 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 Abstracts YSF – Young Scientist Forum 452 FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies response to treatment with NGF and bFGF (Clough and Stefan- is, 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 con- struct lacking the ZSCAN21 binding site exhibited a 40% reduc- tion 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 veri- fied 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 stud- ies 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 Minin 1,2* , A. Lunardi 1,2* , P. Provero 3 , M. Dal Ferro 1,2 , M. Carotti 1,2 , G. Del Sal 1,2 and L. Collavin 1,2 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, 3 Molecular 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 interac- tions of the entire p53 family in man. Using a genome-scale in vi- tro expression cloning approach, we identified 91 previously unreported Dmp53 interactors, considerably expanding the cur- rent Drosophila p53 interactome. Looking for evolutionary con- servation 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 interac- tions. 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 wild- type p53, increased expression of GTPBP4 correlates with reduced patient survival, emphasizing a potential relevance of this regulatory axis in cancer. YSF.26 Mitotic entry regulation by Golgi complex partitioning: a novel mitotic checkpoint R. I. Cervigni 1,2 , M. L. Barretta 1,2 , D. Corda 1 and A. Colanzi 1,2 1 Institute of Protein Biochemistry, National Research Council (IBP-CNR), Naples, Italy, 2 Telethon 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 progres- sive and reversible disassembly of the ribbon structure into dis- persed 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 com- plex 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 frag- mentation 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 Aur- ora-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 Aurora- A activator. To investigate the role of possible candidates we developed a ser- ies 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 Ge ´ ne ´ tique Mole ´ culaire 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 character- ized model pump of Escherichia coli. Starting from combinatorial libraries of small cyclic peptides bio- synthesized in E. coli,1 a selection strategy was designed for iden- tifying 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 centrifuga- tion 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. YSF – Young Scientist Forum Abstracts FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 453 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 regu- lates 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 enlarge- ment, myocyte degeneration and fibrosis. In addition, the trans- genic 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 repre- sents 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 Angio- tensin 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. Evnouchidou 1 , R. Kamal 2 , S. S. Seregin 2 , Y. Goto 3 , M. Tsujimoto 3 , A. Hattori 4 , P. V. Voulgari 5 , A. A. Drosos 5 , A. Amalfitano 2 , I. A. York 2 and E. Stratikos 1 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, 3 Faculty of Pharmaceutical Sciences, Teikyo-Heisei University, Ichihara, Chiba, Japan, 4 Graduate School of Pharmaceutical Sciences, Kyoto University, Department of System Chemotherapy and Molecular Sciences, Sakyo, Kyoto, Japan, 5 Rheumatology Clinic, Department of Internal Medicine, Medical School, University of Ioannina, Ioannina, Greece ER aminopeptidase 1 (ERAP1) customizes antigenic peptide pre- cursors for MHC class I presentation and edits the antigenic pep- 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 dem- onstrates specificity for the whole sequence of its substrates and not only the N-terminus. Recently, coding ERAP1 single nucleo- tide polymorphisms (SNPs) have been associated with an autoim- mune 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 sub- strate 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 sup- porting that ERAP1 allelic composition may affect antigen pro- cessing 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 sub- strate inhibition kinetics. YSF.30 Targeting insulin receptor substrates for destruction as a therapeutic modality for cancers E. Flashner 1,2 , H. Reuveni 1,2 , K. Makedonski 1,2 , A. Shir 1,2 and A. Levitzki 1,2 1 Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel, 2 NovoTyr 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 (tyrphos- tins) 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 inhibi- tory serine phosphorylation and elimination of the IRS1/2 pro- teins. This attribute leads to long lasting inhibition of IGF1R- signal 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 deg- radation is mediated, at least partially, by the proteasome. Inter- estingly, 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. Abstracts YSF – Young Scientist Forum 454 FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies YSF.31 Endocannabinoids and decidualization: CB1 mediates apoptosis through the generation of ceramide and activation of caspase 9 B. M. Fonseca 1,2 , M. Almada 1 , B. Macedo 2 , N. Teixeira 1,2 and G. C. da Silva 1,2 1 Laborato ´ rio de Bioquı ´ mica, Departamento Cie ˆncias Biolo ´ gicas, Faculdade de Farma ´ cia e, 2 IBMC, 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 func- tion and, consequently, conceptus growth/development. How- ever, 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 associ- ated 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]. Cera- mide levels, a lipid second messenger, have been shown to medi- ate 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 decid- ual 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 recep- tors are coupled to the generation of ceramide and the involve- ment 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 activa- tion 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 cleav- ing 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 regu- lated by two networks called FEAR (Cdc Fourteen Early Ana- phase 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 of the model shows the underlying consequences of keeping CycB-dependent Cdk1 activity at a high level. Firstly we ana- lyzed 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 com- prehend 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. Veiga 1 , N. C. Santos 1 , W. Kowalczyk 2 , D. Andreu 2 , A. T. Da Poian 3 and M. Castanho 1 1 Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Physical Biochemistry Unit, 2 Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Barcelona, Spain, 3 Instituto 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 spectro- scopic techniques (Dynamic Light Scattering – DLS, Zeta-Poten- tial, Fluorescence Spectroscopy and Laser Confocal Microscopy). Peptide R and Peptide M, these including respectively the puta- tive 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 pep- tide/oligonucleotides complex formation with high affinity bind- ing 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 parti- tion model for supramolecular complexes). The biological impli- cations 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 periph- eral benzodiazepine receptor (PBR), is a small transmembrane (18 kDa) protein mainly involved in the translocation of choles- terol 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-termi- nal 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- YSF – Young Scientist Forum Abstracts FEBS Journal 278 (Suppl. 1) 446–484 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 455 [...]... on structure parameters ´ ´ Six samples of sulfated cellulose (SC) from Populus tremula wood with different molecular weight (Mr, 1 2–1 9 kDa), polymerization 478 YSF – Young Scientist Forum degree (PD, 5 7–8 7), sulfation degree (SD, 0.5 4–0 .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... interaction FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies YSF – Young Scientist Forum with the excess of polarizing OH) ions This suggestion is also supported by the appearance of intense broad peak within 150 0– 1300/cm corresponding to in-plane bending vibration of the hydroxyl group (145 0–1 310/cm) Furthermore, at pH 9.0... 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 476 YSF – Young Scientist Forum YSF. 101 Protein dysfunction in mitochondrial fatty acid beta oxidation accounts for... indicate an involvement of PARP12 in RNA processing and membrane trafficking FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies YSF – Young Scientist Forum Abstracts YSF. 44 Different membrane interactions of C2 domains YSF. 46 Crystal structures of human sulfotransferase 1A1: from broad to narrow specificity ´ J Guillen, G... amino acids motif GX5EX5[UA]XREX2EEXGU They hydrolyze a variety of nucleoside diphosphate derivatives 468 FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies YSF – Young Scientist Forum YSF. 77 DNA vaccine expressing a-fetoprotein fused with ODC degron: a novel approach for HCC prevention A V Morozov1, A V Timofeev1, V A... cue to locate their prey The cascade of events FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies YSF – Young Scientist Forum involved in indirect defense starts from early signaling events such as intracellular Ca2+ variations, plasma membrane potential (Vm) depolarization and production of reactive oxygen species This... provides insight into how these aSerRS homologues recognize fundamentally different macromolecular substrate FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 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... stroma-leukemic progenitor cells cross talk and deliver new terapeutic targets as well as diagnostic marker FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 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,... 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 462 YSF – Young Scientist Forum YSF. 56 (S12.2.5) Does activation of DNA damage response... Acknowledgements: This work was supported by CNCSISUEFISCDI, PNII- IDEAS grant no.1004/2009, code 1936/2008 FEBS Journal 278 (Suppl 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies 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, . (Suppl. 1) 44 6–4 84 (2011) ª 2011 The Authors Journal compilation ª 2011 Federation of European Biochemical Societies YSF – Young Scientist Forum YSF. 4 Plasma. propagation process. FEMS Yeast Research 10: 87 0–8 84. YSF – Young Scientist Forum Abstracts FEBS Journal 278 (Suppl. 1) 44 6–4 84 (2011) ª 2011 The Authors Journal