Báo cáo khoa học: Invited speakers pot

Invited speakers I01 Please view Plenary lectures I02 Abstract missing, please view Abstract Addendum I03 Aquaporin water channels in health and disease M. Amiry-Moghaddam University of Oslo, Oslo, Norway The water permeability of biological membranes has been a long- standing problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. Peter Agre received Nobel prize in Chemistry in 2003 for this discovery. AQP1 is selectively perme- ated by water driven by osmotic gradients. The atomic structure of human AQP1 has been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single-file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 12 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins) and more than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates. Research in the past decade has shown that aquaporins are not only involved in important physiological processes such as maintenance of body water homeostasis, but are also involved in pathological condi- tions such as nephrogenic diabetes insipidus, cataract, metabolic syndrome, brain edema and epilepsy. I04 Abstract missing, please view Abstract Addendum I05 Electron transfer routes in photosynthetic membranes – impact on biohydrogen production E-M. Aro, P. Zhang, M. Eisenhut, Y. Allahverdiyeva and N. Battchikova University of Turku, Turku, Finland Optimization of electron transfer from water to biohydrogen pro- duction in a model organism Synechocystis sp. PCC 6083 involves several steps. PSII function can be improved by intro- ducing a proper PSII reaction center D1 protein (encoded by the psbA gene family). To this end, an expression of a specific psbA gene that encodes a D1’ protein was detected under anaerobic conditions. We have also demonstrated a novel and crucial func- tion for Flavodiiron (FDP) proteins Flv2 and Flv4 in photopro- tection of PSII. The rate of accumulation of flv2 and flv4 transcripts upon shift of cells from high to low CO 2 is strongly dependent on light intensity. Characterization of FDP inactiva- tion mutants revealed a specific decline in PSII centers and impaired translation of the D1 protein in Dflv2 and Dflv4 when grown at air level CO 2 whereas at high CO 2 the FPDs were dis- pensable. Dflv2 and Dflv4 were also more susceptible to high light induced inhibition of PSII than WT or Dflv1 and Dflv3. Of the four flavodiiron proteins (Flv1-4) in Synechocystis 6803, a physi- ological function of Flv1 and Flv3 is in the Mehler reaction. Up to 30% of electrons derived from water by PSII may be directed to molecular oxygen via Flv1 and Flv3, and thus this route might seriously compete for electrons with the hydrogenase. Besides FDPs, the multiple NDH-1 complexes in cyanobacterial thyla- koid membranes have a crucial role in electron transfer reactions, particularly in cyclic electron transfer around PSI, in respiratory electron transfer and in carbon concentrating mechanisms. More- over, interplay between the FDPs and NDH-1 complexes is dem- onstrated to occur in electron transfer reactions. I06 Global transcriptional regulation of the gut microbiota and its impact on host physiology F. Ba ¨ ckhed University of Gothenburg, Gothenburg, Sweden The adult intestine contains around 100 trillion bacteria, a num- ber 10 times greater than the number of human cells in our body. This complex ecosystem (gut microbiota) has complemented our own genome with several functions that affects human health: modulation of metabolism, development of the immune system, and protection against enteric infections. Recent data have impli- cated the gut microbiota to be involved in obesity, which is asso- ciated with an altered gut microbiota. The gut microbiota can affect host metabolism either directly or indirectly by affecting gene expression. Germ-free mice have provided an important tool to investigate the underlying molecular mechanisms for how the gut microbiota affects host physiology. By using metabolomics and lipidomics we have recently found that the gut microbiota directly affects the serum metabolome and serum, liver, and adi- pose lipidomes. By comparing the transcriptional profile along the length of the gut in germ-free and conventionally raised mice we have demonstrated altered expression of several hormones that are produced by the epithelium. Additional experiments revealed that microbial regulation of gene expression in the small intestine could directly affect host metabolism. Furthermore, we could demonstrate a rapid induction of genes involved in the innate immune system in the epithelium as well as recruitment of immune cells to the mucosa. Taken together the gut microbiota should be considered as an organ by itself that has major effects on host metabolism and physiology, were perturbations may cause or promote disease. I07 Folding and redox processes in the mitochondria L. Banci CERM & Department of Chemistry, University of Florence, Sesto Fiorentino (Florence), Italy A number of proteins undergo their folding through various states that are dependent on the cellular compartment and on compartment-specific protein components. A number of nuclear- encoded proteins, lacking the mitochondrial target sequence, enter mitochondria through transmembrane channels and get trapped in the IMS through oxidative folding processes. These proteins are characterized by disulfide bonds whose formation is combined to protein folding. Disulfide bond formation is kineti- Invited speakers Abstracts FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies 5 cally slow and is catalyzed by a protein, Mia40, which is part of a disulfide relay system. In this system the electron flow goes from Mia40 substrates up to cytochrome c and cytochrome c oxi- dase. Significantly some of the Mia40 substrates are proteins responsible for incorporating copper in cytochrome c oxidase. This behaviour shows how various processes occurring in the same cellular compartment are tightly connected and interlinked, and therefore only a charactrization at ‘system’ level can fully describe functional processes. A few examples of folding pro- cesses will be presented and discussed. I08 New insights into hepatitis C virus replication and persistence R. Bartenschlager Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany Hepatitis C viruses (HCV) comprise the sole genus hepacivirus in the family Flaviviridae. These viruses have in common a single strand RNA genome of positive polarity encoding for a single polyprotein that is cleaved by host and viral proteases. Studies of the HCV replication cycle have become possible by the develop- ment of highly efficient and robust cell culture systems. By using these techniques four molecules (CD81, scavenger receptor class B type I; claudin-1 and occludin) that are essential for infection of hepatocytes have been identified and validated. Moreover, important insights into the biogenesis and architecture of the membranous replication complex induced upon viral infection have been gained. Finally, it was found that HCV assembly occurs in close association with lipid droplets and the host cell machinery required for the synthesis of very-low-density-lipopro- tein (VLDL). As a net result of the assembly reaction, infectious virus particles are formed that are unique in structure and com- position and that resemble most closely LDL. A hallmark of HCV infection is the high rate of persistence (~80%). It was found that the viral serine-type protease NS3 is a key factor that blocks the induction of antiviral cytokines, esp. type 1 interferon (IFN) by proteolytic cleavage of adaptor pro- teins involved in RIG-I and TLR-3 dependent signalling. Apart from blocking innate immunity, HCV overcomes adaptive immune responses by multiple strategies including antigenic vari- ability. Moreover, the tight association of HCV particles with lip- ids appears to impair virus neutralization. Thus HCV utilizes multiple strategies to establish persistence. I09 The billion protein question A. Bateman Wellcome Trust Genome Campus, Hinxton, UK Next generation sequencing is pouring out incredible amounts of data. We are inevitably moving towards the day when we know 1 billion protein sequences. This is both exciting and terrifying. I will discuss ways we might deal with the data deluge. Will we all need to become Bioinformaticians? I10 Salmonella metabolism during infection D. Bumann Biozentrum, University of Basel, Basel, Switzerland Metabolism is an important aspect of Salmonella biology during infection. However, individual metabolic perturbations rarely diminish Salmonella virulence in a mouse typhoid fever model. To understand underlying causes for this large-scale enzyme dis- pensability, we used in silico modeling combined with systematic experimental analysis. Our genome-scale metabolic in silico model correctly predicts virulence phenotypes of more than 92% of 849 experimentally characterized Salmonella mutants. Model- ing and experimental analysis of multiple metabolic mutations revealed a minor impact of network redundancy on overall robustness. However, the host microenvironment provides diverse nutrients that are mainly responsible for extensive Salmonella robustness against perturbation. Interestingly, genome compari- sons suggest that many other pathogens might encounter similar nutritional patterns in their hosts that markedly differ from habi- tats of environmental microbes. I11 Designer cellulosomes: Synthetic multi-enzyme macromolecular complexes E. Bayer Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Cellulosomes are intricate multi-enzyme machines produced by anaerobic cellulolytic microorganisms, designed for efficient decomposition of plant cell wall polysaccharides, notably cellu- lose – the most abundant renewable organic polymer on Earth. The cellulosome complex consists of interlocking, multi-modular, structural and enzymatic subunits, which fit together in a Lego- like arrangement. We have harnessed the molecular logic of the cellulosome components and have developed methodologies to re-tool its precise structural organization. For this purpose, func- tional modular parts of cellulosome subunits from different microorganisms are mixed and matched by recombinant means to produce chimaeric products, which can be fitted together in a controlled manner into novel cellulosome-like structures. The resultant designer cellulosomes are functional, and their cellulo- lytic capacities approach and frequently surpass those of equiva- lent free enzyme systems. Our approach is designed to better understand these intercomponent interactions, to discover how the cellulosome is constructed and how cellulosome architecture contributes to the enhanced synergistic activities of its enzyme components. Knowledge of these interactions provides a broad platform for biotechnological and nanotechnological applica- tions, including prospects for conversion of plant cell wall bio- mass to biofuels – a crucial goal of global importance in the 21st century for all mankind. I12 Pathophysiology of the mitochondrial permeability transition P. Bernardi Biomedical Sciences, University of Padova, Padova, Italy The mitochondrial permeability transition (PT) is a Ca 2+ -depen- dent increase of mitochondrial inner membrane permeability to solutes with molecular masses up to about 1500 Da [Hunter DR et al (1976) J Biol Chem 251: 5069–5077]. Its occurrence is always accompanied by depolarization, while onset of matrix swelling, depletion of matrix pyridine nucleotides, outer membrane rupture and release of intermembrane proteins including cytochrome c depend on the open time. The PT is due to the reversible opening of a high-conductance, voltage-dependent channel in the inner mitochondrial membrane, the PT pore (PTP). In spite of many efforts, its molecular identity remains unknown [reviewed in Ber- nardi P et al (2006) FEBS J 273: 2077–2099]. In this lecture I shall cover the essential aspects of PTP pathophysiology, with specific emphasis on the role of matrix cyclophilin D [Giorgio V et al (2010) Biochim Biophys Acta doi:10.1016/j.bbabio.2009.1 2.006]; Abstracts Invited speakers 6 FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies the mechanism of action of cyclosporin A [Basso E et al (2008) J Biol Chem 283: 26307–26311]; the modulation by the proton elec- trochemical gradient [Bernardi P (1992) J Biol Chem 267: 8834– 8839] and redox effectors [Petronilli V et al (1994) J Biol Chem 269: 16638–16642]; and the consequences of PTP opening as a key to understanding its role in cell dysfunction and death. From this analysis the PTP emerges as a viable target for therapeutic intervention in cancer [Rasola A et al (2010) FEBS Lett doi:10.1016/j.febslet.2010.02.022] and degenerative diseases [Mer- lini L et al (2008) Proc Natl Acad Sci USA 105: 5225–5229], and a highly conserved event across species. I13 Please view Plenary lectures I14 Relationships between structure and functions of a major complement inhibitor C4b-binding protein A. Blom Laboratory Medicine Malmo ¨ , Section of Medical Protein Chemistry, Lund Universty, Malmo ¨ , Sweden The complement system is a vital component of innate immunity and defends host from infections, alerts adaptive immunity and clears the organism from unwanted debris such as dying cells, misfolded proteins and immune complexes. Complement is tightly regulated by a number of soluble and membrane bound inhibitors and disturbances of this regulation are related to dis- eases. C4b-binding protein (C4BP) is a major soluble complement inhibitor composed of seven identical a-chains and a unique b- chain, both of which contain complement control protein (CCP) domains. All subunits are held together at their C-termini via hydrophobic interactions between amphipathic helices and disul- phide bridges. We have localized a number of binding sites for C4BP ligands using recombinant mutants lacking domains, site- directed mutagenesis, modelling and NMR. The binding site for C4b, responsible for inhibition of complement, is localized to CCP1-3 of a-chains and includes mainly positively charged aa. An overlapping site is used by M proteins of Streptococcus pyog- enes, an interaction that is one of the immune evasion mecha- nisms of this pathogen. Overlapping binding sites are also used by heparin and DNA. The C-terminus of a-chains including CCP8 binds in turn some components of extracellular matrix and amyloid. The high affinity (KD 0.2 nM) binding site for antico- agulant protein S is localized to hydrophobic aa on CCP1 of the b-chain. The C4BP-PS complex binds avidly to apoptotic cells allowing their non-inflammatory phagocytosis. I15 Transgenic plastids as expression factories in biotechnology R. Bock Max-Planck-Institute of Molecular Plant Physiology, Potsdam, Germany Genetically modified plants (GM plants) can potentially provide inexpensive production platforms for pharmaceuticals and nutra- ceuticals. With the advent of technologies to alter the genetic information inside plastids (chloroplasts), a new attractive target for genetic engineering has become available to biotechnologists. There are considerable attractions of the plastid genome as a target for the expression of foreign genes. These include (i) the plastids’ potential for high-level foreign protein expression (Zhou et al., 2008; Oey et al., 2009), (ii) the possibility of trans- gene stacking through expression of multiple genes from ope- rons, and (iii) the absence of position effects and epigenetic gene silencing mechanisms (Bock, 2007; Bock and Warzecha 2010). From the biosafety perspective, the major attraction is the exclu- sively or predominantly maternal inheritance of the plastid gen- ome in most crop plants, greatly reducing the risk of uncontrolled pollen spread of transgenes and thus allaying con- cerns over environmental consequences of GM crop cultivation (Ruf et al., 2007). Applications of chloroplast engineering in basic research and biotechnology will depend critically on suc- cess with extending the crop range of chloroplast transformation (Ruf et al., 2001) and the feasibility to express transgenes in non-green plastids (as present in fruits and tubers), which often are less active in gene expression (Kahlau and Bock, 2008). The state of the art in engineering the plastid genome of higher plants will be described and selected applications in two areas of biotechnology will be discussed: metabolic engineering and molecular farming. I16 Structure of chloroplast membrane organization using cryo-electron tomography E. Boekema, R. Kouril and G. Oostergetel University of Groningen, Groningen, Netherlands The thylakoid membrane architecture of chloroplasts was studied by cryo-electron tomography (ET) and single particle electron microscopy (EM). ET reconstructions of isolated, ice-embedded specimens enabled to resolve the features of photosystem II (PSII) in the native membrane of granal stacks and to get a close view of its distribution. 3D analysis of subvolumes containing PSII complexes provided a 3D structure of the PSII core complex at 40 A ˚ resolution. Comparison with a recently proposed pseudo- atomic model of the PSII supercomplex (EMBO Journal 28: 3052) revealed the presence of unknown protein densities right on top of the four peripheral LHCII trimers. The positions of individual PSII complexes were used to fit an entire membrane layer with C2S2M2 supercomplexes. Fitting shows that many supercomplexes must be of smaller size than C2S2M2 super- complexes, to avoid overlap. The characteristic features of PSII enabled assignment of the absolute orientation of individual membranes of granal thylakoid discs and to get a detailed inter- pretation of the membrane folding pattern. This shows that two concentrically folded membranes make a unit of four layers. In that unit, the outer membrane of the grana disc is interrupted on only one side, whereas the inner membrane is almost closed. It will be discussed how this is in line or in contrast with curent models. I17 The genetic landscape of a cell C. Boone University of Toronto, Toronto, ON, Canada A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic inter- actions, generating quantitative genetic interaction profiles for most genes in the budding yeast, Saccharomyces cerevisiae. A network based on quantitative genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets and highly corre- lated profiles delineate specific pathways to define gene function. The global network identifies functional cross connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about Invited speakers Abstracts FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies 7 genetic network hubs in other organisms. An unbiased mapping of the genetic landscape also provides a key for interpretation of chemical-genetic interactions and drug target identification. Finally, the identification of conditional essential genes, which are essential within a specific genetic background, provides a strategy for mapping genetic networks based upon natural varia- tion that distinguishes two individuals. I18 Genomics-enabled approaches for revealing the molecular secrets of marine diatoms C. Bowler Ecole Normale Superieure, Plant Biology, Paris, France Diatoms are eukaryotic photosynthetic microorganisms found throughout marine and freshwater ecosystems that are responsi- ble for around 20% of global primary productivity. A defining feature of diatoms is their ornately patterned silicified cell wall, which display species-specific nanoscale structures. These organ- isms therefore play major roles in global carbon and silicon bio- geochemical cycles. The two available whole genome sequences, from Thalassiosira pseudonana and Phaeodactylum tricornutum, provide a basis for comparative and functional genomics studies of diatoms with other eukaryotes, as well as a foundation for interpreting the ecological success of these organisms in a geno- mic context. Of particular significance is the unprecedented pres- ence of hundreds of genes from bacteria within diatom genomes. The ancient origins of these gene transfers are testified by the finding that more than 300 are found in both diatoms, and many are likely to provide novel possibilities for metabolite manage- ment and for perception of environmental signals. The wide range of genetic resources that have been developed for P. tricor- nutum (e.g. genetic transformation, RNAi, 130 000 ESTs) enable the functions of these and other genes to be assessed using mod- ern molecular methods. As a case in point I will show how gen- ome-enabled resources can reveal how genes from different origins have been recruited to ensure diatom survival in chroni- cally iron-limited regions of the ocean. I19 Structural and functional insights into mitochondrial complex I U. Brandt Johann Wolfgang Goethe-University, Medical School, Frankfurt am Main, Germany Mitochondrial complex I is a membrane integral multiprotein complex composed of 40+ different subunits and a total mass of nearly 1 MDa. As entry point from NADH into the respira- tory chain it serves a central function in energy metabolism and couples electron transfer to ubiquinone with vectorial proton translocation across the inner mitochondrial membrane. The strictly aerobic yeast Yarrowia lipolytica is a powerful genetic system to study complex I. By extensive structure based site- directed mutagenesis we have analyzed the ubiquinone reducing catalytic core of complex I that resides at the interface between the 49-kDa and the PSST subunit of the peripheral arm of com- plex I. In this functionally critical domain the ubiquinone is reduced by cluster N 2 , the last in a chain of seven iron-sulfur clusters. Our results define a likely entry pathway for ubiqui- none leading to a region next to cluster N 2 , where the substrate seems to bind to a fully conserved tyrosine. We could identify residues responsible for the interaction of the isoprenoid side chain of ubiquinone with complex I. Monitoring inhibitor sensi- tivity allowed identification of binding regions for different clas- ses of hydrophobic complex I inhibitors. New evidence on the location of the ubiquinone binding pocket within complex I and the path leading to the site where the hydrophobic substrate gets reduced is presented. The molecular mechanism how complex I couples proton translocation to electron transfer is still unknown. We propose that long range conformational changes drive proton pumping through a two-state stabilization change mechanism involving distinct binding modes of charged ubiqui- none intermediates. I20 Molecular mechanism of photo-adaptation and light entrainment of the circadian clock of Neurospora crassa M. Brunner 1 , E. Malzahn 1 , S. Ciprianidis 1 , T. Schafmeier 1 and K. Kaldi 2 1 Biochemistry Center, Heidelberg University, Heidelberg, Germany, 2 Physiology, Semmelweis University, Budapest, Hungary The transcription factor White Collar Complex (WCC) is a core element of the circadian clock of Neurospora crassa. White Col- lar-1, a subunit of the WCC, is a blue-light photoreceptor required for entrainment of the clock to photoperiods. Activated WCC supports expression of Vivid (VVD), which is a negative regulator of the WCC. WCC and VVD are both required for photoadaptation of Neurospora. We present a molecular mecha- nism how the photocycles of the flavin-binding LOV domains of WC-1 and VVD allow photoadaptation to light intensities over several orders of magnitude. We show that VVD is required for the discrimination between day and night in naturally ambiguous photoperiods with moonlight. I21 Tuning the proton pumping stoichiometry in cytochrome c oxidase by single mutations P. Brzezinski Stockholm University, Stockholm, Sweden Cytochrome c oxidase (CytcO) is a multisubunit membrane- bound enzyme, which catalyzes the oxidation of cytochrome c and reduction of molecular oxygen to water. Part of the free energy available from this reaction is used to pump one proton across the membrane per electron transferred to oxygen. This pumping stoichiometry of 1 H + /e - can be modulated by intro- duction of single-site mutations, as far away as ~25 A ˚ from the catalytic site, to obtain essentially any stoichiometry in the range 0–1. In many cases these mutations leave the intramolecular elec- tron and proton-transfer rates unaffected. Understanding the molecular mechanism underlying the partial or full uncoupling of proton pumping from oxygen reduction is important for under- standing the molecular design of the proton-pumping machinery in the respiratory oxidases. One key feature of all ‘uncoupled mutants’ studied to date is that the pK a of an internal proton donor, Glu286, located deeply within a proton pathway used for transfer of pumped and sub- strate protons, is either increased or decreased (from 9.4 in the wild-type oxidase). Because the Glu residue is found near the cat- alytic site as well as near a putative exit pathway for pumped protons, the altered pK a presumably reflects changes in the local structural environment of the residue, which are important for controlling the rates and trajectories of proton transfer to these sites. These results establish a link between specific structural changes in the proton pump and, its thermodynamic and kinetic properties thereby offering mechanistic insights into the molecu- lar design of the proton-pumping machinery of the oxidases. Abstracts Invited speakers 8 FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies I22 High resolution quantitative mass spectrometry for analysis of proteomes and PTMs C. Choudhary Department of Proteomics, University of Copenhagen, The NNF Center for Protein Research, Copenhagen, Denmark Proteins are fundamental constituents of all living organisms. Mammalian genomes contain thousands of protein coding genes (20 000 in human), and by alternative splicing of the mRNA, and variance in gene sequences, they may encode many more protein products. A great majority of proteins in eukaryotic cells are modified by different posttranslational modification (PTMs). PTMs are key constituents of cell signaling networks, compris- ing some of the most sophisticated control switches in the cell. Cells use various regulatory mechanisms such as dynamic regu- lation of protein expression, PTMs, and protein-protein interac- tions, to orchestrate cellular processes and to adapt to environmental clues. Given the vast complexity and their all essential role in controlling biology of living cells, analysis of proteins and PTMs is one of the most exiting areas of research in biology. Recent developments in high-resolution mass spectrometry (MS), computational proteomics, and optimized PTM-enrich- ment strategies have greatly increased the depth of proteome and PTM coverage by MS. Global phosphorylation analysis in mammalian cells can routinely identify more than 10 000 phos- phorylation sites in a single experiment. Large-scale analysis of other PTMs such as lysine acetylation, ubiquitylation, and meth- ylation are also becoming amenable to this technique. Quantita- tive MS screens can provide unbiased, systems-wide information about changes in protein expression, protein modifications, and protein-protein interactions. I will discuss recent developments in the quantitative MS, and highlight their pioneering applica- tions in quantification of proteomes and posttranslational modi- fications. I23 The interplay between phosphorylation and ubiquitination in regulating the innate immune system P. Cohen University of Dundee, Medical Research Council Protein Phosphorylation Unit, Dundee, UK Infection by bacteria and viruses triggers cells of the innate immune system to produce pro-inflammatory cytokines and inter- ferons that mount the responses to fight the invading pathogens. However these defence mechanisms are a double-edged sword because the uncontrolled production of these substances causes chronic inflammatory and autoimmune diseases. This talk will focus on the signaling pathways downstream of Toll-Interleukin Receptors. The engagement of these receptors by their agonists triggers the formation of Lys63-linked polyubiquitin chains and polyubiquitylated proteins, which then recruit and activate the key protein kinases that drive the production of pro-inflamma- tory cytokines and type 1 interferons. The mechanisms that initi- ate the formation of Lys63-linked polyubiquitin chains and the proteins that interact with them (NEMO, OPTN, ABIN1 and ABIN2) will be discussed. Finally, the characterization of mice that express an ABIN1 mutant unable to bind to Lys63-linked polyubiquitin chains will be described and a model that accounts for the striking phenotype displayed by these knock-in mice will be presented. I24 Functional interaction between chromatin remodelers and non-coding RNA’s D. Corona Dipartimento di Biologia Cellulare e dello Sviluppo, Universita’ degli Studi di Palermo & Dulbecco Telethon Institute, Palermo, Italy The Hsr-x gene is developmentally expressed in almost all cells types of D. melanogaster and is one of the most strongly induced heat shock genes in flies. The Hsr-x locus encodes multiple non- coding RNAs (ncRNA); the large nuclear species (Hsr-x-n) is essential for the assembly and organization of hnRNP-containing omega speckles. These special nuclear compartments are thought to play essential roles in the storage/sequestration of members of hnRNP family and other proteins playing important roles in RNA maturation. ISWI is an evolutionarily conserved ATP- dependent chromatin remodeler playing essential roles in chro- mosome condensation, gene expression and DNA replication and is also involved in a variety of nuclear functions including telo- mere silencing, stem cell self-renewal, neural morphogenesis and epigenetic reprogramming. Using an in vivo assay to identify fac- tors that antagonize ISWI activity, we recovered a genetic inter- action between ISWI and Hsr-x. Loss of Hsr-x function results in a strong suppression of eye morphology and chromosome con- densation defects caused by loss of ISWI activity. Moreover, the organization of the omega speckles in ISWI mutant cells is pro- foundly altered when compared to wild type cells. Interestingly, immuno-FRISH analysis revealed a significant number of sites in the nucleus where the chromatin-bound ISWI protein overlaps the omega speckles-associated Hsr-x ncRNA. Remarkably, RNAimmunoprecipitation assays, conducted in salivary glands extracts, revealed a physical interaction between ISWI and Hsr-x ncRNA. I25 The bacterium Listeria monocytogenes: a brilliant cell biologist and an insidious biochemist P. Cossart Unite ´ des Interactions Bacte ´ ries-Cellules, Institut Pasteur, Paris, France Listeria monocytogenes is an ubiquitous bacterium that can con- taminate food products and induce several forms of a disease called listeriosis, an infection which is mortal in 30% of the cases. Infection is mainly due to the capacity of the organism to live and replicate in both phagocytic and non phagocytic cells. It is also due to the property of the organism to be able to cross three host barriers during infection, the intestinal barrier, the blood brain barrier and in pregnant women the materno-fetal barrier. A series of approaches have highlighted that this bacte- rium has evolved amazing mechanisms to switch from saprohytic life to virulence, to escape early innate host immune defense, to invade non phagocytic cells, to spread from cell to cell and to cross host barriers. We will focus the talk on recent results that have highlighted new concepts in endocytosis/phagocytosis and revealed. Invited speakers Abstracts FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies 9 I26 Molecular characterization of estrogen receptor signaling in breast cancer cells C. Zhao 1 , H. Gao 1 , N. Gustafsson 1 , Z. Papoutsi 1 , S. Jaffrey 1 , J-A ˚ . Gustafsson 2 , J-A ˚ . Gustafsson 1 and K. Dahlman-Wright 1 1 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, 2 Department of Biology and Biochemistry, University of Houston, Houston, TX, USA Breast cancer is a common malignancy worldwide. Although the 5-yr survival rate has increased with advances in detection and treatment, many breast cancer patients still die from metastatic disease. Therefore, more effective methods for diagnosis, classifi- cation, treatment and individualized treatment are greatly needed. Estrogen, acting via estrogen receptors (ERs), ERa and ERb, has been well documented to play a critical role in the eti- ology and progression of breast cancer. The introduction of anti-estrogens such as tamoxifen for the treatment of breast can- cer constitutes a milestone in the treatment of this disease. How- ever, many tumors are not responsive to this treatment or become resistant over the cause of the treatment. Our research is focused on understanding aspects of estrogen signaling at the molecular level including how estrogen signaling can be modu- lated by other pathways than ligand-binding. To increase our understanding of the molecular mechanism of estrogen signaling in breast cancer cells, we determined the global DNA-binding profile ERa and ERb and showed that there were differences in the DNA-binding profiles between these two receptors including differences with regard evolutionary conservation, distance to transcription start sites and binding site characteristics. We have shown that ERa signaling and proliferation can be inhibited by the ERb variant ERb2 through proteasome-dependent degrada- tion of ERa and enhanced by the RBCK1 protein, the latter might involve direct regulation of the ERa promoter. More recently we have focused on crosstalk between AP-1 and ERb signaling showing that inhibition of AP-1 signaling decreases ERb DNA-binding and signaling. I27 On the role of protons and O 2 partial pressure in water oxidation by photosystem II H. Dau, I. Zaharieva, M. Haumann, J. Wichmann, A. Grundmeier, A. Klauss and L. Gerencser Freie University Berlin, FB Physik, Berlin, Germany In plants and cyanobacteria, two water molecules are split into four energized electrons (reducing equivalents), four protons and one dioxygen molecule. This process is driven by light and facili- tated by a manganese-calcium complex bound to protein subunits of photosystem II (PSII), the latter being an impressive cofactor- protein complex embedded in the thylakoid membrane separating the lumen and stroma compartment. Experimental results on the ‘backpressure’ by the reaction products ‘proton’ (low pH at the lumen side of PSII) and ‘oxygen’ (high O 2 partial pressure) are presented. The results relate to the mechanism of water oxidation in PSII, specifically to the spatio-temporal coordination of elec- tron and proton transfer at the donor side of PSII. Implications for the function of PSII in intact organisms also will be discussed. I28 Exocytosis, endocytosis and lysosomal targeting of the Aquaporin-2 water channel: an interplay of phosphorylation and ubiquitination P. Deen Physiology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands Regulation of a proper water homeostasis is essential for terres- trial animals to survive. This regulation is mainly achieved by drinking and modulation of reabsorption of water from pro-urine, of which the latter is under control of the anti-diuretic hormone vasopressin. In states of hypernatremia or hypovolemia, vasopres- sin is released from the pituitary, binds its type-2 receptor in the kidney collecting duct and induces the apical surface expression of aquaporin-2 water channels upon activation of a cAMP signaling cascade. When AVP levels drop, but also with additional vaso- pressin-counteracting hormones, AQP2 is internalized for recy- cling or degradation. Using polarized cells or renal material, we found that forskolin/vasopressin increases Ser256 phosphoryla- tion (pS256) of AQP2, which is essential for its translocation to the apical membrane. This translocation was reversed upon removal of forskolin or co-incubation with vasopressin-counter- acting hormones, which did not involve pS256 dephosphorylation, but essentially involved a transient increase in AQP2 K63-linked ubiquitination with 2–3 ubiquitin moieties on Lys270 only in AQP2. This ubiquitination was an essential process, as AQP2- K270R, which cannot be ubiquitinated, was impaired in its endo- cytosis. AQP2 stably fused to ubiquitin (AQP2-Ub) localized to lysosomes. Ubiquitin-dependent endocytosis of AQP2 was fol- lowed by AQP2 phosphorylation at S261, which seems to stabilize AQP2 ubiquitination and endocytosis. Following endocytosis, AQP2 is targeted for degradation to multivesicular bodies and lysosomes, which involves interaction with the MIT1 domain of LIP5, which is part of the ESCRTIII complex. I29 Ubiquitin-mediated regulation of NF-jB activation and autophagy I. Dikic Institute of Biochemistry II, Goethe University Frankfurt, Germany An increasing number of distinct functions have been assigned to different types of ubiquitin modifications (mono ubiquitin versus ubiquitin chains). In these processes Ub acts as a signalling com- ponent able to trigger molecular events in cells. Structural studies have revealed significant differences between ubiquitin chains of Lys48, Lys63 and linear linkages. We have recently shown that the UBAN domain of NF-jB essential modulator (NEMO) binds specifically to linear Ub chains and that this step is essential for NF-jB activation. These findings explain the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency. More recently, we have identi- fied novel components of the linear ubiquitin-dependent pathways that control the NF-jB and apoptotic pathways downstream of TNF receptors. Removal of harmful protein aggregates, damaged organelles and microbes is mediated by autophagy, a process by which the cell sequesters cytosolic cargo and delivers it for degra- dation by the lysosome. The involvement of ubiquitin in selective autophagy is becoming more evident: autophagic clearance of protein aggregates requires specific autophagy receptors. Identifi- cation of p62/SQSTM1 and NBR1 as autophagy receptors, which simultaneously bind both ubiquitin and autophagy-specific ubiqu- itin-like modifiers, LC3/GABARAP, has provided a molecular Abstracts Invited speakers 10 FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies link between ubiquitination and autophagy. The molecular details of selective autophagy suitable for clearance of various cargoes, ranging from ubiquitinated protein aggregates to membrane- bound organelles and microbes will be discussed. I30 Abstract missing, please view Abstract Addendum I31 The Structural basis for ion conduction and gating in pentameric ligand-gated ion channels R. Dutzler Department of Biochemistry, University of Zurich, Zurich, Switzerland The pentameric ligand-gated ion channels (pLGICs) constitute a family of neurotransmitter receptors that are key players in the control of electric signaling at chemical synapses. The family codes for a conserved scaffold of channel proteins that open in response to the binding of neurotransmitter molecules. We have determined the X-ray structures of two prokaryotic family mem- bers from the bacterium Erwinia chrysanthemi (ELIC) at 3.3 nm resolution [1] and from the bacterium Gloeobacter violaceus (GLIC) at 3.1 nm resolution [2]. Both proteins form cation selec- tive channels and bear most of the structural hallmarks of the family including the N-terminal extracellular ligand-binding domain and the four helices of the pore domain. Despite the overall similarity, both structures adopt distinct conformations of the ion conduction pathway. The structure of ELIC shows a non-conductive state with rings of hydrophobic residues at the extracellular side of the pore preventing ion permeation. This hydrophobic barrier has opened in the structure of GLIC to a funnel shaped pore, where a ring of conserved glutamate residues at the intracellular constriction creates an ion-coordination site. GLIC is thus believed to represent a conducting conformation of the channel. In combination, both structures suggest a novel gat- ing mechanism for pentameric ligand-gated ion channels where channel opening proceeds by a change in the tilt of the pore- forming helices. The structures thus provide a first detailed view into how a pLGIC may open and selectively conduct ions. References: 1. Hilf RJC, Dutzler R. Nature 2008; 452: 375–379. 2. Hilf RJC, Dutzler R. Nature 2009; 457: 115–118. I32 Lessens from the pH-sensitivity of the rate of peptidyl transfer from P-site to A-site tRNA on the translating ribosome M. Ehrenberg 1 , M. Johansson 1 , I. Kaweng 1 , P. Strazwski 2 , M. Pavlov 1 and J. Aqvist 1 1 Uppsala University, Dept of Cell and Molecular Biology, Uppsala, Sweden, 2 Universite ´ Claude Bernard, Lyon 1, Institut de Chimie et Biochimie Mole ´ culaires, Lyon, France We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNAfMet to the aa-tRNAs Phe-tRNAPhe, Ala-tRNAAla, Gly-tRNAGly, Pro-tRNAPro, Asn-tRNAAsn and Ile-tRNAIle, selected to cover a large range of intrinsic pKa- values for the a -amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and had different apparent pKa-values, defined as the pH-value at which the rate was half maximal. The apparent pKa-values were downshifted in relation to those of the aa-tRNAs in solution. Gly-tRNAGly had the smallest (about 0.25 pH unit), while Ile-tRNAIle and Ala- tRNAAla had the largest (about 1.5 pH unit) downshifts. These down shifts correlate strongly with molecular dynamics (MD) estimates of the down shifts in pKa-values of the alpha-amino groups of these aa-tRNAs upon A-site binding. Our data demon- strate the chemistry of peptide bond formation, rather than accommodation, to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and strongly suggest rate limiting chemistry for all aa-tRNAs. I33 Lipidomics in health & disease K. Ekroos Zora Biosciences, Espoo, Finland Lipidomics is defined as the systems-level analysis of lipid species, their abundance, biological activity, as well as sub-cellular locali- zation and tissue distribution. Technological advances in mass spectrometry and associated method development have produced lipidomic techniques capable in characterizing and quantifying hundreds of molecular lipid species directly in total lipid extracts. Moreover, the high detection precision at the molecular lipid level prevents false-positive identifications, a necessity for quality lipid research and enables studies requiring GLP. The identifica- tion of lipid metabolic dysfunctions responsible for disease pathology also requires the coupling of customized bioinformatic tools to the analytical chemistry of lipidomics. Molecular lipids are the window to meaningful biological insight and a prerequi- site for piecing together the molecular mechanisms of health and disease processes. Finally, molecular lipidomics is a valuable addition to the toolkits used both in biomarker discovery and in the identification of new drug targets. I34 Probing intracellular kinetics at the level of single molecules B. English, A. Sanamrad, H. Vasili and J. Elf Uppsala university, Uppsala, Sweden I will present our resent advancements in tracking individual freely diffusing fluorescent protein molecules at high time resolu- tion in the cytoplasm of bacterial cells. In vivo tracking of indi- vidual proteins molecules makes it possible to study kinetics high time resolution without synchronizing the population of mole- cules. For example by monitoring the kinetics of the response mediator RelA we have developed a single molecule assay to study stress response and starvation at the level of individual bacteria. The RelA protein binds to a small fraction of ribo- somes, where it synthesizes the global transcriptional regulator ppGpp in response to amino acids deprivation. This the ppGpp molecule binds to the RNAP and rapidly reprograms the cell for the new environment, in what is called the stringent response. While Escherichia coli contains on average about 100 RelA mole- cules and 10 000 ribosomes, using a photo-activatable fluorescent probe we can activate only a few fluorescent molecules per cell at any given time and track them at high time resolution. When the cell grows exponentially, RelA trajectories closely resemble trajec- tories of fluorescently tagged ribosomal proteins (D~0.4 lm 2 /s as compared to D~0.3 lm 2 /s for ribsomes). After nutritional down- shift, RelA binding kinetics changes rapidly and the protein diffuses very fast (D~3.5 lm 2 /s) as if it only binds to ribsomes transiently. The assay has made it possible to study the rapid and transient stringent response in individual cell as well as the heterogeneity in the stress response over the population. Invited speakers Abstracts FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies 11 I35 Human brown adipose tissue S. Enerba ¨ ck University of Gothenburg, Go ¨ teborg, Sweden Scarcity of food and cold weather have accompanied us and con- stituted serious threats to our survival for the entire duration of our species’ history. Fortunately, two tissues have been selected by nature to help us deal with these difficulties: white adipose tis- sue (WAT) and brown adipose tissue (BAT). While WAT helps us compensate for fluctuating availability of energy, BAT keeps us warm – at least during our first days of extra-uterine life. As will be discussed, it is most likely of importance to us for longer than that. It is only very recently that, by having an adequate food supply and modern housing, we have circumvented the problems associated with famine and cold weather – at least in some parts of the world – and thus become less dependent on WAT and BAT for our survival. This has occurred to the extent that new dangers have been created. A sedentary inactive lifestyle and a wealth of calories have generated a cluster of obesity- related health problems known as the metabolic syndrome or syndrome X. Which in turn is associated with a positive energy balance; here BAT can play a role by increasing energy expendi- ture. BAT is only present in mammals and is estimated to have emerged some 150 million years ago. BAT is unique in the sense that brown adipocytes can uncouple respiration from ATP syn- thesis. A role for metabolically active BAT in the normal physiol- ogy of healthy adults will be discussed as well as potential therapeutic implications. I36 Abstract missing, please view Abstract Addendum I37 Mechanisms of initiation of DNA replication in human mitochondria M. Falkenberg Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden Mitochondrial DNA is replicated by a unique enzymatic machin- ery, which is distinct from the replication apparatus used for copying the nuclear genome. In combination, DNA polymerase gamma and TWINKLE DNA helicase form a processive replica- tion machinery, which can use dsDNA as template to synthesize single-stranded DNA molecules and addition of the mitochon- drial ssDNA-binding protein stimulates the reaction further. Recently, we have studied the mechanisms of origin-specific initi- ation of lagging-strand DNA synthesis. We found that the mito- chondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we could faithfully reconstitute OriL- dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure and at this stage, POLRMT initiates primer synthesis from the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase gamma, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mito- chondria. In ongoing studies, we are trying to understand how mtDNA replication is initiated at the leading-strand origin of mtDNA replication, OriH. We will present data suggesting a new mecha- nism for transcription termination and primer formation in human mitochondria. I38 Conformational changes seen in crystal structures of oxidized, reduced and mutant forms of cytochrome c oxidase: clues to coupling and gating mechanism? S. Ferguson-Miller, L. Qin, J. Liu, C. Hiser and F. Li Michigan State University, Biochemistry and Molecular Biology, East Lansing, MI, USA There is increasing recognition that altered mitochondrial meta- bolism and energy production is involved in many disease states, particularly those associated with neurological dysfunction, aging and obesity. Cytochrome c oxidase is a critical controlling enzyme in the mitochondrion, but despite intensive study, impor- tant aspects of its mechanism and regulation remain elusive. We study a bacterial homolog of the mammalian mitochondrial enzyme, which has a simpler structure, is readily mutated, and is amenable to crystallographic analysis. Our recent high-resolution crystal structures provide new insights into its ligand binding behavior (Qin et al, Biochemistry 2008; 47: 9931) and new evi- dence of redox-linked conformational change (Qin et al, Bio- chemistry 2009; 48: 5121). An exciting aspect of these results is that the changes seen upon reduction of the enzyme suggest a conformationally-controlled gating mechanism that has not pre- viously been considered. Another interesting finding is that lipid and steroid binding sites are conserved from bacterial through to mammalian oxidases, suggesting functional importance. Crystal structures of mutant forms in proton uptake pathways show minimal alteration in structure except for missing key water molecules, but reveal similar redox-linked conformational changes as wildtype. Spectral changes in crystals during x-ray irradiation reveal evidence of a strained configuration in the fro- zen state. The importance of conformational change and lipidic ligands in the mechanism and regulation of cytochrome oxidase is a focus of our current studies. (NIH GM26916; MSU REF 03- 016). I39 Nature’s way to exploit dihydrogen as an alternative fuel B. Friedrich Humboldt University Berlin, Institute of Biology/Microbiology, Berlin, Germany Hydrogen, an attractive microbial energy source, is released into the anoxic environment by fermentation where it gets immedi- ately consumed by anaerobic organisms. Both hydrogen evolu- tion and hydrogen consumption are catalyzed by metal-cofactor containing hydrogenases. In order to use these catalysts for effi- cient production of sunlight-driven hydrogen from water, a num- ber of requirements have to be met. Particular challenges are: (i) tight coupling of the electron flow between the algal or cyanobac- terial photosystems and the hydrogenase protein and (ii) toler- ance of the hydrogenase towards oxygen that unavoidably escapes during the water splitting process. The majority of hy- drogenases are sensitive to oxygen and either irreversibly dam- aged or inactivated by oxygen. A few [NiFe] hydrogenases, resident in aerobic hydrogen-oxidizers, tolerate ambient oxygen during catalysis. The molecular background of oxygen tolerance is diverse and may depend on the access of oxygen to the active site, on the interplay between various metal cofactors, their Abstracts Invited speakers 12 FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies protein environment and their redox potentials. Insights into these mechanisms open new strategies for genetic engineering of an optimal hydrogenase catalyst suitable for biotechnological applications. Some account of this work has been published in a recent review article. (O. Lenz, M. Ludwig, T. Schubert, I. Bu ¨ r- stel, S. Ganskow, T. Goris, A. Schwarze, B. Friedrich. H 2 con- version in the presence of O 2 as performed by the membrane- bound [NiFe]-hydrogenase of Ralstonia eutropha. ChemPhy- sChem (2010) DOI:10.1002/cphc.200901002.) I40 Abstract missing, please view Abstract Addendum I41 From hot to cool and more for less: new developments for structural biology E. Garman Biochemistry, University of Oxford, Oxford, UK Structural biology relies on X-ray crystallography to provide much of the three dimensional information on macromolecules that informs biological function. To enable problems not previ- ously accessible to structure solution to be tackled, improved methods must be developed. A notable example of this has been the progress in finding protocols to cryocool protein crystals prior to data 100K collection to reduce the rate of radiation damage by around a factor of 70 compared to that at room tem- perature (RT): from hot to cool and more for less. Radiation damage to the sample is an inherent problem when utilising ion- ising X-radiation in MX, and it is now known that radiation damage can also be a limiting factor for MX at 100K. Following our measurement of 30 MGy (1 Gy = 1 J/kg energy absorbed) for the experimental dose limit for 100K protein crystals, we tried to determine a limit for RT samples. The unexpected results will be presented. Current ongoing methods investigations that will be described include studies of 100K and RT radiation damage in macromolecular crystals in order to inform both our understanding and putative mitigation strategies, and trace ele- mental analysis of liquid and crystalline proteins using micro- PIXE (particle induced X-ray emission), allowing determination of their stoichiometric ratio to an accuracy of between 10% and 20%. I42 Transport through nuclear pore complexes S. Frey 1 ,B.Hu ¨ lsmann 1 , A. Labokha 1 , C. Ader 2 , M. Baldus 2 and D. Go ¨ rlich 1 1 Max Planck Institute for Biophysical Chemistry, Cellular Logistics, Go ¨ ttingen, Germany, 2 Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, Netherlands The permeability barrier of nuclear pore complexes (NPCs) is a passive and yet highly efficient sorting device that controls all exchange between nucleus and cytoplasm. It suppresses the flux of inert macromolecules >30 kDa, but also allows rapid passage of even very large cargoes, provided these are bound to appropri- ate nuclear transport receptors (NTRs). FG-repeat domains bind NTRs during facilitated NPC passage and constitute the crucial elements of this barrier. They are essential for viability and com- prise up to 50 repeat units. Each unit contains a hydrophobic cluster, typically of the sequence FG, FxFG or GLFG, sur- rounded by more hydrophilic spacer sequences. We observed that FG-repeat domains form FG-hydrogels. These gels are fascinat- ing materials that display permeability properties very similar to those of authentic NPCs, allowing an up to 20,000-fold faster entry of large NTRdcargo complexes as compared to the cargoes alone. While supporting massive importin- or exportin-mediated cargo influx, such gels remain firm barriers towards inert objects that lack nuclear transport signals. This indicates that FG-hydro- gels reseal immediately behind a translocating species and thus possesses self-healing properties. The presentation will further address the following questions: What is the molecular and struc- tural basis of barrier formation? Why do the meshes of the bar- rier open at least 100 times faster in the immediate vicinity of a nuclear transport receptor than elsewhere in the gel? How do FG repeat domains behave on a nanoscopic scale, i.e. in authentic NPCs? I43 Abstract missing, please view Abstract Addendum I44 High-resolution imaging of neurotransmitter receptors: learning from a single molecule L. Groc CNRS – Universite ´ Bordeaux, Bordeaux, France In the nervous system, the trafficking of neurotransmitter receptor in and out synapses has emerged as a key process through which synapses adapt their strength in response to salient environmental cues. Although numerous molecules and signalling cascades have been identified over the last decades, our current understanding of the molecular dynamics that sustain synaptic adaptations is still rather limited. The membrane insertion and endocytosis of neurotransmitter receptors mostly regulate the number of surface receptor in various membrane compartments. Using single mole- cule and particle tracking it recently emerged that surface neuro- transmitter receptors are highly dynamic. Such observation required the development of new experimental approaches to spe- cifically label and track receptor with appropriate time- and space-resolutions. Physiologically, it unravelled that the lateral diffusion of glutamatergic receptors, which mediate most excit- atory drive in the brain, plays a major role in regulating receptor trafficking to and from synaptic sites as well as the short- and long-term adaptations of excitatory synapses. These develop- ments, i.e. how studying single surface receptor at the nano-scale level provided fundamental knowledge about the synaptic and neuronal plasticity, will be discussed in this presentation. I45 Mechanistic insights into complement activation and regulation P. Gros Crystal and Structural Chemistry, Utrecht University, Utrecht, Netherlands The complement system is a crucial part of the innate immunity in mammals. This system is formed by ~30 plasma proteins and cell-surface receptors, which enable the host to clear invading pathogens and altered host cells, while protecting healthy host tissue. Through structural studies we have revealed the molecular mechanisms responsible for the amplification and regulation of the central steps in the complement pathways. Structures of the central complement component C3 (1641 res.) and its activated form C3b revealed intricate domain arrangements and marked conformational changes that lead to covalent labelling by C3b, which mark target cells for immune clearance [1,2]. Activation is performed by labile protease complexes, i.e. C3 convertases, Invited speakers Abstracts FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies 13 which are formed on the target cell surfaces. We determined structures of the pro-enzyme factor B [3], the C3 pro-convertase [4] and the active short-lived (half-life time ~90 s) C3 convertase [5]. Furthermore, we determined structures of C3b in complex with complement regulators that protect host cells from comple- ment activation [6]. Together our data provide comprehensive insights into the formation, activity, specificity and regulation of the central C3 convertase, which cleaves C3 into the active C3b and thereby amplifies the complement response in immune clear- ance. References: 1. Janssen et al. Nature 2005; 437: 505–511. 2. Janssen et al. Nature 2006; 444: 213–216. 3. Milder et al. Nature SMB 2007; 14: 224–228. 4. Janssen et al. EMBO J 2009; 28: 2469–2478. 5. Rooijakkers, Wu et al. Na Immun 2009; 10: 721–727. 6. Wu et al. Nat Immun 2009; 10: 728–733. I46 Please view Plenary lectures I47 Systematic analysis of dynamic signaling modules by quantitative mass spectrometry M. Gstaiger, M. Varjosalo, A. Wepf, T. Glatter, O. Rinner, R. Aebersold and A. van Drogen ETH, IMSB, ETH Ho ¨ nggerberg, Zu ¨ rich, Switzerland Biological processes such as metabolism and signal transduction emerge from dynamic networks of interacting protein complexes. Affinity purification combined with mass spectrometry (AP-MS) represents the most widely used approach for studying protein complexes under near physiological conditions. However compre- hensive analysis of protein complexes that constitute entire sig- naling systems in human cells is limited by a number of experimental challenges. These limitations are primarily linked to the lack of time resolved quantitative MS data on transient sig- nalling dependent protein-protein interactions, problems in iden- tifying the true protein complex composition from AP-MS data and the low throughput of existing AP-MS workflows. We have introduced a combined computational and mass spectrometric framework for the quantitative analysis of changes in protein complex composition and protein modifications. We will also report on a recently developed strategy for the systematic analy- sis of human interaction proteomes at increased through put. When combined these approaches mark a significant step for- ward towards deciphering dynamic networks of human signalling complexes. Recent applications of these methods for the analysis of cellular signaling modules that control of cell growth will be discussed. I48 Abstract missing, please view Abstract Addendum I49 The dynamic amyloid landscape P. Hammarstrom IFM-Department of Chemistry, Linkoping University, Linkoping, Sweden We investigate conformational heterogeneity and templated con- formational conversion of amyloidogenic proteins associated with human diseases. We study the human prion protein, the amyloid beta peptide and transthyretin which are all linked to protein deposition in the form of amyloid in vivo. There is ample evi- dence that different point mutations in these proteins dictate the disease phenotype. For the human prion protein around 20 dif- ferent point mutations and two SNPs (rendering single residue substitutions) have been described which all present different dis- ease phenotypes. The same phenomenon holds true for transthy- retin (>100 mutations have been identified) and for amyloid beta which also extensively varies in peptide chain length, depending on processing. Our goal is to understand the prerequi- sites for amyloid fibril formation and conformational stability of both the native structure and the misfolded conformations. We have found that several self-assembled forms of these proteins can interconvert emphasizing that these are dynamic structures with rather shallow barriers between them. References: 1. Almstedt K et al. Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates. Prion 2009; 3(4): 224–235. 2. So ¨ rgjerd K. et al. Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture. Biochem Biophys Res Commun 2008; 377(4):1072–1078. 3. Nilsson KP et al. Imaging distinct conformational states of amy- loid-beta fibrils in Alzheimer’s disease using novel luminescent probes. ACS Chem Biol 2007; 2(8):553–560. I50 Abstract missing, please view Abstract Addendum I51 Extensive crosstalk between O-GlcNAcylation and phosphorylation: a new paradigm for cellular signaling? G. Hart Biological Chemistry, Johns Hopkins University School of Medi- cine, Baltimore, MD, USA The cycling of O-linked b-D-N-acetylglucosamine (O-GlcNAc) at serine or threonine moieties is abundant on nuclear and cyto- plasmic proteins, where it serves as a nutrient/stress sensor to regulate signaling, transcription and cellular metabolism. Several studies have shown that a modest increase in global O-GlcNAcy- lation affects phosphosite occupancy at nearly every actively cycling phosphorylation site. Recently, in collaboration with Don Hunt’s group at the University of Virginia, we developed a chemico-enzymatic photochemical enrichment method, combined with electron transfer dissociation (ETD) mass spectrometry, that allows, for the first time, detection of O-GlcNAc site occupancy on nucleocytplasmic proteins at a level of sensitivity comparable to that possible for phosphorylation. Using this method to study signaling processes that regulate cell division, we found that the crosstalk between site specific phosphorylation and O-GlcNAcy- lation is surprisingly extensive, with nearly every actively cycling phosphorylation site affected by altered O-GlcNAcylation. Major known signaling cascades that regulate cell division are strikingly affected by a modest change in O-GlcNAcylation. Our studies suggest that O-GlcNAcylation generally modulates many signal- ing cascades and phosphate-mediated molecular switches to allow them to be highly responsive to nutrients and stress. Sup- ported by NIH R01 CA42486 and DK61671. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHUSOM. I52 Abstract missing, please view Abstract Addendum Abstracts Invited speakers 14 FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies [...]... activating a multitude of potential signaling pathways, only one of which is the cAMP signaling pathway To analyze PGE2 signaling through the signal network elicited by activation of the four EP receptors in lymphoid cells, we introduced a multi-pronged strategy, combining temporal quantitative phosphoproteomics and phospho flow cytometry to describe PGE2 signaling networks 32 Invited speakers I140 Molecular... and which allow us to test the parameters affecting the initiation of the virological synapse between cells Another extension to conventional fluorescence microscopy, 3D structured illumination 16 Invited speakers super-resolution microscopy, further allows us to dissect the spatial structure of these adhesive synapses between cells Combined, these biophysical imaging and manipulation tools permit us... of osteoclasts, premature osteoclasts incubated on bone particles FEBS Journal 277 (Suppl 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies Invited speakers with the appropriate growth factors and co-cultures with breast cancer cells were also evaluated ZA effectively inhibits cell proliferation, in vitro invasion and migration of cancer cells... pattern between adipocytes and macrophages In lean subjects, genes related to adipocyte metabolic pathways are highly expressed whereas genes of macrophage inflammatory pathways show low expression An 18 Invited speakers opposite pattern is seen in obese patients with metabolic syndrome suggesting a crosstalk between adipocytes and macrophages within adipose tissues which is related to the extent of fat mass... conditions and in vivo and protects the fungus against external stresses FEBS Journal 277 (Suppl 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies Invited speakers Our current knowledge of the three-dimensional structural organisation of all these constitutive cell wall polysaccharides in A fumigatus and the enzymes that are responsible for their synthesis,... to its target promoters We propose that CK2 antagonizes CCA1 regulatory activity to modulate the transcriptional network controlling morning and evening phase-specific expression in Arabidopsis 20 Invited speakers I79 Abstract missing, please view Abstract Addendum I80 Abstract missing, please view Abstract Addendum I81 Trans-splicing is a regulated and is master regulator of trypanosome gene expression... phosphorylations are self-inflicted and the phosphorylated residues FEBS Journal 277 (Suppl 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies Invited speakers Abstracts participate in the reaction mechanism of the enzymes in question But there also seem to be phosphorylation events that do not have any effect on the target proteins Is that only... single-molecule studies that examine replisome action during lagging strand synthesis and the consequences for the leading strand polymerase I91 Reconstruction and multi-scale modeling of lipid networks Invited speakers sis is fundamental to maintain health, and lipid defects are central to the pathogenesis of important and devastating diseases The spatial complexity of lipid metabolism presents a challenge... plays a vital role in many cellular processes including signalling, 22 FEBS Journal 277 (Suppl 1) 5–36 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies Invited speakers homeostasis, nutrient uptake and in defining the interactions of cells with its surrounding It is also the route by which many pathogens gain access to the cell The AP2 adaptor complex (a/... collection and dissemination of the assays Finally, to overcome the bottlenecks of SRM assay development, we introduced a method based on the use of unpurified synthetic peptide libraries, that 24 Invited speakers allows for the high-throughput and low-cost generation of validated SRM assays (>100 SRM assays/h) for any set of proteins or proteome of interest The approach was used to develop a complete . Invited speakers I01 Please view Plenary lectures I02 Abstract missing, please view. formation is combined to protein folding. Disulfide bond formation is kineti- Invited speakers Abstracts FEBS Journal 277 (Suppl. 1) 5–36 (2010) ª 2010 The Authors