Báo cáo khoa học: Abstract P1 – FEBS Datta Plenary Lectureship Award P1-001 Peptide bond formation, cotranslational folding and antibiotics synergism docx

Abstract P1 – FEBS Datta Plenary Lectureship Award P1-001 Peptide bond formation, cotranslational folding and antibiotics synergism A. Yonath Structural Biology, Weizmann Inst., Rehovot, Israel. E-mail: ada.yonath@weizmann.ac.il Ribosomes position their substrate at stereochemistry suitable for peptide bond formation, and promote substrate-mediated cata- lysis. The linkage between substrate orientation, dominated by remote interactions, and a sizable symmetrical region identified in all known ribosome structures indicates a guided rotatory motion of aminoacylated-tRNAs along a ribosomal path leadings to the advance of nascent peptides into the protein exit tunnel at an extended conformation. The symmetry related region can transfer intra-ribosomal signals between remote locations, since it con- nects all ribosomal functionally sites. These included the deco- ding and peptide-bond-formation sites; the protein exit tunnel, the tRNA entrance and exit environments and the protein exit tunnel entrance. The symmetry relates RNA backbone and nucle- otides orientation, but not sequence homology. Thus, suggesting that ribosomes evolved by gene-fusion and demonstrates the superiority of the functional requirements over sequence conser- vation. The protein exit tunnel acts as a dynamic functional entity capable of taking part in nascent protein elongation, dis- crimination, arrest and partial protein folding. Initial steps in chaperon-aided cotranslational folding are associated with signifi- cant mobility of both the bacterial trigger factor and a ribosomal protein at the tunnel opening. Similarly, major conformational alterations, induced by ribosomal recycling factor play a key role in the termination steps of protein biosyntheses. Comparative analysis of antibiotics binding modes to a eubacterial pathogen model and an archaeal sharing properties with eukaryotes showed that despite the overall conservation of the ribosome, phylogenetic and conformational variations in antibiotics binding pocket allow their selectivity, thus facilitating their therapeutical usage. P2 – 50 th Anniversary IUBMB Lecture P2-001 Protein misfolding and human disease: what we have learned from 50 years of protein science C. M. Dobson Department of Chemistry, University of Cambridge, Cambridge, UK. E-mail: cmd44@cam.ac.uk Proteins are the most abundant molecules in biology, other than water, and enable or regulate all the chemical processes on which life depends. Over the past 50 years our knowledge and understanding of these complex molecules has increased out of all recognition. The methods of X-ray diffraction, NMR spectr- oscopy and electron microscopy, coupled with theoretical tech- niques such as molecular dynamics simulations, have together given us deep insight into their structures and properties. In addition, a very wide range of biophysical and biochemical studies, particularly exploiting the power of protein engineering techniques to probe the roles of individual amino acid side chains, has revealed many of the intimate mechanistic details of how individual protein molecules are able to exert their specific functions. In addition to the question of how the structures of proteins are related to their functions, an additional question emerged as soon as the first structures of proteins were solved. This question concerns the manner in which these fascinating and intricate structures are attained by polypeptide chains fol- lowing biosynthesis in the cell as an essentially unstructured chain of amino acids. This process of protein folding is of par- ticular significance not just because it links gene sequences to biological activity, but also because it represents perhaps the most universal example of biomolecular self-assembly, a phe- nomenon on which all life depends. In recent years very consid- erable progress has been made towards understanding the fundamental basis of protein folding through the concerted application of a series of experimental approaches, notably the variety of biophysical techniques along with the methods of protein engineering, coupled with theoretical and computational approaches. On the basis of these studies, the outline of a uni- versal and comprehensive mechanism of folding is emerging, and indeed is beginning to shed light on the way in which the amino acid sequence encodes the protein fold. The development of techniques to study protein folding has also resulted in major advances in our ability to define the structures and dynamics of proteins in states other than the native ones. This topic is of considerable interest because these states are increasingly recog- nized as being coupled to many biological processes ranging from molecular trafficking to cell signalling and the regulation of the cell cycle. In addition, however, it has also become evi- dent that the failure to fold correctly, or to remain correctly folded, is the origin of a wide variety of human disorders ran- ging from Alzheimer’s disease to type II diabetes. The study of such misfolding events and their consequences has been made possible by the adaptation of techniques developed to study the normal structural and folding characteristics of proteins. As well as shedding light on the nature of individual diseases, these studies have provided evidence for underlying generic aspects of protein misfolding and its consequences. These conclusions are now providing insight into the origins of these diseases, why they are becoming epidemic in many parts of the world, and how they might be treated on a rational basis. In addition they raise a series of fascinating issues about the underlying nature of biological molecules and the driving forces of molecular evo- lution. This lecture will attempt to bring these threads together to give an overview of our present understanding of the nature of protein molecules and how it has emerged over the past half century. 1 References 1. Dobson CM. Protein folding and misfolding. Nature 2003; 426: 884–890. 2. Dobson CM. In the footsteps of alchemists. Science 2004; 304: 1259–1262. 3. Dobson CM. Chemical space and biology. Nature 2004; 432: 824–828. P3 – Theodor Bu ¨ cher Lecture and Medal P3-001 Metabolomics, modelling and machine learning in systems biology; understanding complex systems using genetic programming to produce simple interpretable rules. The Theodor Bu ¨ cher Lecture and Medal D. B. Kell Chemistry, University of Manchester, Manchester, Lancs, UK. E-mail: dbk@manchester.ac.uk Progress in Systems Biology – or in ‘understanding complex sys- tems’ – depends on new technology [1–3], computational assist- ance [4] and new philosophy [5], but probably not in that order (pace [6]). Some developments include all three [7, 8]. References 1. Kell DB. Metabolomics and systems biology: making sense of the soup. Curr Op Microbiol 2004; 7: 296–307. 2. Goodacre R, Vaidyanathan S, Dunn WB, Harrigan GG & Kell DB Metabolomics by numbers: acquiring and under- standing global metabolite data. Trends Biotechnol 2004; 22: 245–252. 3. O’Hagan S, Dunn WB, Brown M, Knowles JD & Kell DB. Closed-loop, multiobjective optimisation of analytical instru- mentation: gas-chromatography- time-of-flight mass spectro- metry of the metabolomes of human serum and of yeast fermentations. Anal Chem 2005; 77: 290–303. 4. Ihekwaba A, Broomhead DS, Grimley R, Benson N & Kell DB. Sensitivity analysis of parameters controlling oscillatory signalling in the NF-kappaB pathway: the roles of IKK and IkappaBalpha. Systems Biology 2004; 1: 93–103. 5. Kell DB & Oliver SG. Here is the evidence, now what is the hypothesis? The complementary roles of inductive and hypoth- esis-driven science in the post-genomic era. Bioessays 2004; 26: 99–105. 6. Brenner S. Nature June 5, 1980. 7. King RD, Whelan KE, Jones FM, Reiser PGK, Bryant CH, Muggleton SH, Kell DB & Oliver SG. Functional genomic hypothesis generation and experimentation by a robot scien- tist. Nature 2004; 427: 247–252. 8. Nelson DE, Ihekwaba A, Kell DB & White MRH. Oscillations in NF-kappaB signalling control the dynamics of target gene expression. Science 2004; 306: 704–708. P4 – PABMB Plenary Lecture P4-001 Structure-based antibiotic design on the bacterial membrane N. C. J. Strynadka Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC Canada. E-mail: natalie@byron.biochem.ubc.ca Antibiotic resistance has become a major clinical problem worldwide. Our lab is interested in the structure-based design of inhibitors which target antibiotic resistance mechanisms or novel targets essential to bacterial pathogenesis. The key determinant of broad spectrum b-lactam resistance in Methicillin superbug strains is the membrane spanning penicillin binding protein 2a (PBP2a), a transpeptidase that is required to produce peptide cross links that give the bacterial cell wall its necessary strength and rigidity. Due to its low affinity for b-lactams, PBP2a pro- vides cross-linking transpeptidase activity at b-lactam concentra- tions which inhibit the other cell-wall transpeptidases normally produced by S. aureus and other pathogenic bacteria. The crys- tal structures of native PBP2a from MRSA has been deter- mined to 1.8 A ˚ resolution as well as acyl-enzyme complexes with various b-lactam antibiotic substrates. An analysis of the PBP2a active site reveals the structural basis of its broad spec- trum resistance to the 50 clinically utilized b-lactam antibiot- ics, and identifies features important for high affinity binding. This information has been used in structure-based inhibitor design strategies aiming to combat MRSA resistance. In terms of novel targets, our laboratory has made significant progress on the structural elucidation of the Type III secretion apparatus (TTSS) common to many Gram-negative pathogens. The TTSS allows for the specific injection of bacterial proteins into human host cells, where they mediate their pathogenic effects. Our laboratory has provided the first high resolution structures of TTSS proteins including the EspA translocation tube that spans from the bacteria OM to host membrane, the outer membrane secretin pilot protein and the inner membrane polymeric ring that is thought to act as the initial ‘platform’ upon which the other Type III components assemble. These structures provide the foundation for understanding the molecular details of this fascinating pathogenic process as well as for the design of novel anti-microbials. Abstracts 2 P5 – Sir Hans Krebs Lecture and Medal P5-001 The epigenome in the context of the post- genomic era T. Jenuwein Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Vienna, Austria. E-mail: jenuwein@imp.univie.ac.at The last years were highlighted by the landmark description of the genomes of many model organisms, including the human gen- ome. These ‘genome projects’ have shown that more complex eukaryotic organisms (e.g. mammals) have a much bigger gen- ome than less complex eukaryotes (e.g. flies), although the increased ‘biocomplexity’ is not reflected by an equivalent increase in the number of protein coding genes. Mechanisms other than DNA sequence information have been adopted during evolution to better index and regulate the various developmental programmes and key regulatory processes, such as gene expres- sion, chromosome segregation and cell division of eukaryotic genomes. In the nuclei of almost all eukaryotic cells, genomic DNA is highly folded and compacted with histone and non- histone proteins in a dynamic polymer called chromatin. The discoveries that nucleosome remodelling machines and histone- modifying enzymes organize chromatin into accessible (euchro- matic) and inaccessible (heterochromatic) configurations reveal epigenetic mechanisms that considerably extend the information potential of the genetic code. Thus, one genome can generate many – epigenomes – as the fertilized egg progresses through development and translates its information into a multitude of cell fates. These epigenetic mechanisms are crucial for the func- tion of most, if not all, chromatin-templated processes and link alterations in the chromatin structure to gene regulation, X inac- tivation, chromosome organization and genome stability. The implications of epigenetic research for human biology and dis- ease, including stem cells, cancer and aging are far-reaching and will form a modern foundation to explore the chromatin template in a ‘post-genomic’ era. P6 – Special Plenary Lecture P6-001 Molecular mechanisms of bacterial swimming and tumbling K. Namba Protonic NanoMachine Group, Graduate School of Frontier Biosciences, Osaka, Suita, Osaka Japan. E-mail: keiichi@fbs.osaka-u.ac.jp The bacterial flagellum is made of a rotary motor and a long helical filament by means of which bacteria swim. The flagellar motor rotates at around 300 Hz and drives the rapid rotation of each flagellum to propel the cell movements. The long helical fil- ament, which is a tubular structure with a diameter of about 20 nm, is made of a single protein flagellin. The filament switches between left- and right-handed helical forms in response to the twisting force produced by reversal of the motor rotation, allow- ing bacteria to alternate their swimming pattern between running and tumbling for taxis. The flagellum also has a short, highly curved segment called hook, which connects the motor and the helical propeller. Its bending flexibility makes it work as a nano- scale universal joint, while the filament is relatively more rigid to function as a propeller. A very short segment made of proteins HAP1 and HAP3 connects these two mechanically distinct struc- tures. The flagellum is constructed by self-assembly of proteins translocated from the cytoplasm through the narrow central channel to the distal end of the growing structure, where one of three different cap complexes is attached to help efficient self- assembly of particular proteins that need to be assembled at each specific stage of the assembly process. We have solved most part of the structures in the cell exterior by X-ray crystallography, fiber diffraction and electron cryomicroscopy. All these structures present interesting implications for the function of each molecule and subcomplex, demonstrating the importance of dual nature of protein molecules, dynamic flexibility and subatomic level preci- sion. P7 – EMBO Lecture P7-001 Dynamics of spliceosome components in the living cell nucleus M. Carmo-Fonseca Institute of Molecular Medicine, University of Lisbon, Lisbon, Portugal. E-mail: carmo.fonseca@fm.ul.pt The spliceosome is a dynamic RNA-protein macromolecular machine that is responsible for the splicing of intronic sequences from pre-mRNA. The spliceosome undergoes major structural changes during the splicing reaction and its components must be recycled for each new round of splicing. Although the spliceosome cycle has been extensively studied at the molecular level, very little is known about the dynamics of spliceosome components in vivo. We are using Fluorescence Recovery After Photobleaching (FRAP) to analyze the mobility and kinetic be- havior of spliceosome components in the nucleus of living human cells. In addition we are performing Acceptor Photo- bleaching Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) to visualize and spatially map the interactions between the splicing factors within the nucleoplasm (where splicing takes place) and in nuc- lear speckles (where splicing components accumulate when not engaged in splicing). Our results support the view that splicing factors assemble onto pre-spliceosome complexes localized in nuclear speckles. Abstracts 3 P8 – EMBO Young Investigator Lecture P8-001 Intracellular signaling in neutrophils and osteoclasts A. Mo ´ csai 1 and C. A. Lowell 2 1 Department of Physiology, Semmelweis University, Budapest, Hungary, 2 Department of Lab. Medicine, University of California, San Francisco, CA, USA. E-mail: mocsai@puskin.sote.hu Immunoreceptors (BCR, TCR, Fc-receptors) signal by a common mechanism whereby receptor-associated ITAM-bearing adaptors become phosphorylated by Src-family kinases and then recruit the Syk tyrosine kinase through its SH2-domains. We found that neutrophils lacking Src-family kinases, the ITAM-bearing adapt- ers DAP12 and FcRc or the Syk tyrosine kinase failed to initiate integrin-induced antimicrobial responses. Phosphorylation of DAP12 and FcRc was defective in cells lacking Src-family kinas- es, and the phosphorylation of Syk was absent both in Src-family deficient and DAP12 –/– FcRc –/– cells. We also found severe oste- opetrosis in mice lacking both DAP12 and FcRc. DAP12 –/– FcRc –/– double and Syk –/– single mutant bone marrow cells failed to differentiate into mature osteoclasts and did not resorb bone. DAP12 and FcRc were constitutively phosphorylated in wild type but not Src-family deficient osteoclasts. In turn, DAP12 and FcRc were required for the constitutive phosphory- lation of Syk. Retroviral expression of wild type Syk or DAP12 was able to restore osteoclast development and function in the relevant knockout background, but this functional reconstitution was abrogated by loss-of-function point mutations in the C-ter- minal SH2 domain of Syk or in the two tyrosines within the DAP12 ITAM motif. These results suggest that integrin-mediated antimicrobial responses of neutrophils and the development and function of osteoclasts require an immunoreceptor-like signaling mechanism, whereby Src-family mediated phosphorylation of DAP12 and FcRc leads to a phospho-ITAM dependent activa- tion of Syk, which is in turn required for downstream signaling and functional responses. Abstracts 4 . Abstract P1 – FEBS Datta Plenary Lectureship Award P1- 001 Peptide bond formation, cotranslational folding and antibiotics synergism A. Yonath Structural. lacking both DAP12 and FcRc. DAP12 –/ – FcRc –/ – double and Syk –/ – single mutant bone marrow cells failed to differentiate into mature osteoclasts and did not