Báo cáo khoa học: PLENARY LECTURES pdf

PLENARY LECTURES EMBO LECTURE I01 Design principles of biological circuits U. Alon Weizmann Institute of Science, Rehovot, Israel Evolution tinkers with odds and ends, but the resulting systems look like circuits designed according to good engineering princi- ples. This talk will discuss several design principles, and how they can be used to make sense of complex transcription and signal- ling networks in cells. SPECIAL LECTURES I13 Telomeres and telomerase E. Blackburn University of California, San Francisco, CA, USA Telomeres, by protecting and stabilizing the ends of chromo- somes, play a vital role in ensuring genomic stability. Each telo- mere consists of simple repeated DNA sequences, which bind cellular protein factors and make a cap, thus securing the chro- mosome end. Without telomeric DNA and its specialized modes of replicating, chromosome ends dwindle away, eventually caus- ing cells to cease dividing. For humans to live a long life, this erosion of telomeres is counteracted by the cellular enzyme telo- merase, which replenishes telomeres by elongating and protecting them. The emerging understanding of telomeres and telomerase, in both normal and transformed cells, holds promise for improv- ing health and combatting cancer. While telomerase is present in many normal cells in human adults, it is often there at only low levels. Throughout human life a minimal level of telomerase is required for replenishment of tissues, including the immune sys- tem. Telomerase is influenced by genetic and non-genetic factors. Telomerase activity in immune system cells is affected by chronic psychological stress, and inadequate telomere maintenance is associated with a variety of major risk factors for diseases includ- ing cancer and cardiovascular disease. Within the setting of malignant cancer cells – a very different setting from normal cel- lular contexts – telomerase is hyperactive and promotes cancer. We have explored exploiting the high telomerase activity of can- cer cells for anti-cancer approaches. The challenge is to develop the emerging molecular and cellular information about telomer- ase into rational cancer therapies and prevention strategies. FEBS/EMBO WISE AWARD LECTURE I46 Wisely chosen paths: regulation of ribosomal RNA synthesis I. Grummt German Cancer Research Center, DKFZ-ZMBH Alliance, Molecular Biology of the Cell II, Heidelberg, Germany All cells, from prokaryotes to vertebrates, synthesize vast amounts of ribosomal RNA to produce 1–2 million ribosomes per cell cycle, which are required to maintain the protein syn- thetic capacity of the daughter cells. In recent years, considerable progress has been made towards the elucidation of both the basic principles of transcriptional regulation and the pathways that adapt cellular rRNA synthesis to metabolic activity, a process essential to understanding the molecular mechanisms that link nucleolar activity to cell growth, proliferation and apoptosis. I will survey our present knowledge of the highly coordinated net- works that regulate transcription by RNA polymerase I, coordi- nating rRNA gene transcription and ribosome production with environmental cues and will discuss the epigenetic mechanisms that control the chromatin structure and transcriptional activity. Particular focus will be made on the role of non-coding RNA in DNA methylation and transcriptional silencing. IUBMB LECTURE I73 Protein folding and inheritance of environmentally acquired characteristics S. Lindquist Whitehead Institute for Biomedical Research, Cambridge, MA, USA Protein folding mechanisms exert a profound effect on how geno- types are translated into phenotypes. Heat shock protein (Hsp) 90 is an abundant molecular chaperone that promotes the folding and maturation of a particularly interesting group of clients: metastable signal transducers that are key regulators of a broad spectrum of biological processes. We have helped to define 2 mechanisms by which Hsp90 influences the acquisition of new phenotypes. First, by robustly maintaining signaling pathways, Hsp90 buffers the effects of mutations, allowing the storage of cryptic genetic varia- tion that is released by stress. In this case, when the Hsp90 buffer is compromised, new traits appear. Second, Hsp90 potentiates effects of genetic variation, allowing new mutations to produce immediate phenotypes. In this case, when Hsp90 function is compromised, new traits are lost. Such changes can be assimilated so that they are maintained under stress. In recent work we have mapped hundreds of traits in diverse strains of yeast, identified specific polymorph- isms involved, and established that Hsp90 has played a broad role in shaping current genomes. Another line of work involves protein- based hereditary elements, prions, which produce changes in phe- notype through a heritable, self-perpetuating change in protein conformation. Prions can be cured or induced by stress, creating heritable new phenotypes that depend upon the genetic variation present in the organism. Hsp90 and prions provide plausible Plenary lectures Abstracts FEBS Journal 277 (Suppl. 1) 1–4 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemi cal Societies 1 mechanisms for allowing genetic diversity and fluctuating environ- ments to fuel the pace of evolutionary change. BU ¨ CHER LECTURE I94 Analyses of pleistocene genomes S. Pa ¨ a ¨ bo Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany A number of technical developments – including high-throughput DNA sequencing – have made it possible to reliably analyze entire genomes that are tens of thousands of years old. We are using these approaches to study the genomes of early human forms, such as Neandertals, who are the closest evolutionary rela- tives of present-day humans. Thus, for any definition of what sets fully anatomically modern humans apart from other hominin forms, the relevant comparison is to Neandertals. I will discuss methodological issues relevant for the analysis of ancient ge- nomes. I will present a draft sequence of the Neandertal genome composed of over 3 billion nucleotides from three individuals. Finally, I will discuss the prospect of analyzing genomes of other early human, such as Cro Magnon, in the future. CLOSING LECTURE I107 What we have learned from structures of the ribosome V. Ramakrishnan MRC Laboratory of Molecular Biology, Cambridge, United Kingdom The determination of the atomic structures of the ribosomal sub- units in 2000 revolutionized the field of translation. I will describe our contributions to this field by focusing first on the structure of the 30S subunit and its complexes, and then on high- resolution structures of functional complexes of the entire ribo- some. These studies have led to an understanding of the struc- tural basis of the accuracy of translation, and how recognition of the codon by the correct tRNA leads to incorporation of an amino acid to the growing polypeptide chain. KREBS LECTURE I136 How a lipid mediates tumour suppression H. Stenmark Centre for Cancer Biomedicine, University of Oslo, Institute for Cancer Research, the Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, NORWAY Phosphorylated derivatives of the membrane lipid phosphatidyl- inositol (PtdIns), known as phosphoinositides, regulate mem- brane-proximal cellular processes by recruiting specific protein effectors involved in cell signalling, membrane trafficking and cytoskeletal dynamics. Two phosphoinositides that are generated through the activities of distinct phosphoinositide 3-kinases (PI3Ks) are of special interest in cancer research. PtdIns(3,4,5)P3, generated by class I PI3Ks, functions as tumour promotors by recruiting effectors involved in cell survival, proliferation, growth and motility. Conversely, there is evidence that PtdIns(3)P, gener- ated by class III PI3K, functions in tumour suppression. Three subunits of the class III PI3K complex (Beclin 1, UVRAG and Bif1) have been independently identified as tumour suppressors, and their mechanism of action in this context has been proposed to entail activation of autophagy, a catabolic pathway that is thought to function tumour suppressive by scavenging damaged organelles that would otherwise produce reactive oxygen species that cause DNA instability. We have recently obtained evidence for three additional functions of PtdIns(3)P that might contribute to its tumour suppressors activity. One such mechanism involves ligand-mediated downregulation of growth factor receptors medi- ated by the endosomal sorting complex required for transport (ESCRT) machinery. Another potential tumour suppressor mech- anism of PtdIns(3)P is mediated by its participation in signalling pathways. The third and most recent potential tumour suppressor mechanism involves the regulation of cytokinesis, the final stage of cell division. Elucidation of the mechanisms of tumour sup- pression mediated by class III PI3K and PtdIns(3)P will identify novel Achilles heels of the cells defence against tumourigenesis and will be useful in the search for prognostic and diagnostic bio- markers in cancer. OPENING LECTURE I142 Breeding and building molecules to spy on cells and tumors X. Shu, V. Lev-Ram, E. Olson, T. Aguilera, T. Jiang, M. Whitney, J. Crisp, T. Deerinck, M. Ellisman, L. Ellies, Q. Nguyen and R. Tsien University California San Diego, La Jolla, CA, USA We are developing genetically encoded tags for electron micros- copy (EM). Arabidopsis phototropin, a photoreceptor containing flavin mononucleotide (FMN), can be engineered into a small (106-residue) Singlet Oxygen Generator (miniSOG), which effi- ciently generates 1 O 2 upon blue light illumination. 1 O 2 polymer- izes diaminobenzidine into an osmiophilic deposit, enabling correlative EM with nanometer spatial resolution. The closely related cell-adhesion molecules SynCAM1 and SynCAM2, sepa- rately fused to miniSOG, predominantly localize respectively to the pre-synaptic and post-synaptic sides of mammalian CNS syn- apses. MiniSOG may do for EM what GFP did for optical microscopy. For clinical applications, we need synthetic mole- cules with novel amplifying mechanisms for homing to diseased tissues. Activatable cell penetrating peptides (ACPPs) are poly- cationic cell penetrating peptides (CPPs) whose cellular uptake is minimized by a polyanionic inhibitory domain and then restored upon proteolysis of the peptide linker connecting the polyanionic and polycationic domains. Invasive tumors secrete matrix metal- loproteinases, which cut the linker and cause amplified retention in tissues and uptake into cells. ACPPs on dendrimers labeled with Cy5 and Gd-DOTA enable whole body magnetic resonance imaging followed by fluorescence-guided surgery. Such fluores- cence guidance improves tumor-free survival in two animal mod- els. Chemotherapeutic drugs also gain efficacy when targeted to the tumor by ACPPs. Thrombin-cleavable ACPPs accumulate in atherosclerotic plaques. Separately, we have developed fluores- cent peptides that light up peripheral nerves to show surgeons where not to cut. Abstracts Plenary lectures 2 FEBS Journal 277 (Suppl. 1) 1–4 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies SPECIAL LECTURES I153 The mechanism and regulation of F-ATPases J. E. Walker Medical Research Council Mitochondrial Biology Unit, Cambridge, UK More than 25 high-resolution structures of mitochondrial F1- ATPase have been determined to date. Comparison of all of the structures with each other, and examination of lattice contacts in the crystals used to solve each structure show that neither the conformations adopted by the catalytic subunits nor the occu- pancy of those subunits by nucleotides is influenced by lattice contacts. Therefore, the structures interpreted as representing ground and transition states depict the structures of intermediates in the catalytic cycle. In the ground state two of the catalytic sites are attached by nucleotides and the third site is unoccupied, whereas in the transition state, nucleotides occupy all three cata- lytic sites. Two recent structures, one of yeast F1-ATpase inhib- ited with yeast inhibitor protein, IF1, the other of the enzyme crystallized in the presence of phosphonate, appear to represent post-hydrolysis pre-product release intermediates. The current structures occupy about 20 ° of each of the three 120 ° steps in a complete 360 ° catalytic cycle. The lecture will discuss strategies for accessing structures that represent the missing part of the cat- alytic cycle. It will also discuss the different regulatory mechan- isms of F-ATPases from mitochondria, chloroplasts and bacteria, by the inhibitor protein, by a redox switch, and possibly by the binding of ATP to the e-subunit respectively. DATTA LECTURE I160 Gene/Environment influence on skeletal muscle insulin sensitivity in Type 2 diabetic patients J. R. Zierath Karolinska Institutet, Stockholm, Sweden Skeletal muscle is an important site of insulin-mediated glucose uptake and defects in this insulin target tissue precede the mani- festation of Type 2 diabetes. Our central hypothesis is that acti- vation of insulin-independent pathways to glucose transport in skeletal muscle may overcome the profound impairment in whole body glucose homeostasis associated with Type 2 diabetes. Inten- sive research efforts have been directed towards understanding the regulation of insulin-dependent and insulin-independent path- ways governing glucose metabolism and the factors causing insu- lin resistance in Type 2 diabetes. Physical exercise/muscle contraction elicits an insulin-independent increase in glucose transport and perturbation of this pathway may bypass defective insulin signaling. To date, the exercise-responsive signaling mole- cules governing glucose metabolism in skeletal muscle are largely unknown. Epigenetic modification through DNA methylation is implicated in metabolic disease and may play a role in the mechanism by which environmental factors influence metabolic responses in diabetes. Using whole genome promoter methylation analysis of skeletal muscle from normal glucose tolerant and Type 2 diabetic subjects, we identified cytosine hypermethylation of Peroxisome Proliferator-Activated Receptor c Coactivator-1 a (PGC-1a) in diabetic subjects. Methylation levels were negatively correlated with PGC-1a mRNA and mitochondrial DNA (mtDNA). Bisulfite sequencing revealed that the highest propor- tion of cytosine methylation within PGC-1a was found within non-CpG nucleotides. Non-CpG methylation was acutely increased in human myotubes by exposure to tumor necrosis fac- tor-a (TNF-a) or free fatty acids, but not insulin or glucose. Selective silencing of the DNA methyltransferase 3B (DNMT3B), but not DNMT1 or DNMT3A, prevented palmitate-induced non- CpG methylation of PGC-1a and decreased mtDNA and PGC- 1a mRNA. We provide evidence for PGC-1a hypermethylation, concomitant with reduced mitochondrial content in Type 2 dia- betic patients, and link DNMT3B to the acute fatty-acid induced non-CpG methylation of PGC-1a promoter. By identifying the molecular mechanisms controlling insulin sensitivity, future devel- opment of pharmacological and physiological (exercise and diet) intervention strategies aimed to improve glucose homeostasis may be possible. FEBS Journal Prize Lecture Generation and characterization of a monoclonal antibody as the first specific inhibitor of human NTPDase3 M. N. Munkonda 1 , M. Fausther 1 , V. V. Ivanenkov 2 , J. Pelletier 1 , A. Tremblay 1 ,B.Ku ¨ nzli 3 , T. L. Kirley 2 and J. Se ´ vigny 1 1 Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Que ´ bec, Universite ´ Laval, Que ´ bec, QC, Canada, 2 Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA, 3 Department of General Surgery, Universita ¨ tMu ¨ nchen, Munich, Germany Background: The study and potential therapeutic modulation of purinergic signaling is hindered due to the lack of specific inhibitors for nucleoside triphosphate diphosphohydrolases (NTPDases), the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural ele- ments that affect enzymatic activity and could be used as targets for inhibitors. Here we report the generation of the first inhibi- tory monoclonal antibodies specific for human NTPDase3. Methods: Antibody specificity was assessed by Western blot, flow cytometry and immuno(cyto/histo)chemistry. Inhibition assays were performed using recombinant NTPDases from pro- tein extracts and intact transfected cells, and human pancreas sections. Epitope determination was performed using mutants of human NTPDase3 and chemical cleavage by cysteine substitution mutagenesis. Results: The monoclonal antibodies inhibit exclusively human NTPDase3 by about 75%. Antibody recognition is attenuated by denaturation with SDS, and abolished by reduction with DTT, indicating the significance of the native conformation and the disulfide bonds in NTPDase3 for epitope recognition. The SDS- resistant parts of the epitope are located in two fragments, Leu220-Cys347 and Cys347-Pro485, both required for antibody binding. In the first fragment, Ser297 is likely to directly interact with antibody. In the second fragment, the principal part of the epitope is likely located near the disulfide bond Cys399-Cys422. Conclusion: We identified the first inhibitory antibodies of an NTPDase and partially defined their epitope. These monoclonal antibodies are likely to be a valuable tool for both biochemical studies and for modulation of purinergic processes controlled by NTPDase3, including insulin secretion by pancreatic islet cells. Plenary lectures Abstracts FEBS Journal 277 (Suppl. 1) 1–4 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies 3 FEBS Letters Young Group Leader Lecture Protein splicing in Cis and Trans: a versatile tool for structural biology and post- translational modulations of protein functions A. S. Aranko, J. S. Oeemig, G. Volkmann and H. Iwai Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland Protein splicing is a remarkable post-translational modification of proteins in which an intervening sequence, termed intein, cata- lyzes self-excision from a host protein and concomitant ligation of the two flanking polypeptide chains, termed exteins. Protein splicing could occur not only in cis but also in trans by catalyzing ligation of two polypeptide chains via split intein fragments. The concerted reactions catalyzed by inteins have opened a multitude of biotechnological applications, such as protein purification, protein cyclization, site-specific labeling, protein semi-synthesis, and segmental isotopic labeling for Nuclear Magnetic Resonance (NMR) studies of larger proteins and intact proteins with repeat- ing sequences. Protein trans-splicing by split intein fragments has great potential as protein ligases for engineering proteins in vitro as well as in living cells. To expand the applications of protein trans-splicing significantly for structural analysis of larger multi- domain proteins by NMR spectroscopy and post-translational control of protein functions in living cells, we have advanced the protein ligation approaches by split inteins through the discovery of robust inteins, NMR analysis of the structure and dynamics of inteins, NMR-based rational design of novel split inteins, and better understanding of protein splicing mechanism to optimize protein ligation conditions. The advanced protein ligation tech- nology based on protein trans-splicing could become an indispen- sable and powerful tool not only in structural biology but also in cell biology. Abstracts Plenary lectures 4 FEBS Journal 277 (Suppl. 1) 1–4 (2010) ª 2010 The Authors Journal compilation ª 2010 Federation of European Biochemical Societies . PLENARY LECTURES EMBO LECTURE I01 Design principles of biological circuits U. Alon Weizmann. genetic variation present in the organism. Hsp90 and prions provide plausible Plenary lectures Abstracts FEBS Journal 277 (Suppl. 1) 1–4 (2010) ª 2010 The Authors