Báo cáo khoa học: PLENARY LECTURES DATTA LECTURE pdf

PLENARY LECTURES DATTA LECTURE PL 1 Drug discovery in the p53 pathway D. Lane Division of Molecular Medicince, College of Life Sciences Medical Sciences Institute, Edinburgh, UK Small molecules that activate the transcriptional function of wild type p53 have proved active as anti-cancer drugs in preclinical models and are now entering clinical trial. The complexity of the upstream p53 signaling pathway offers many targets for the devel- opment of such activators and the action of Mdm2 inhibitors, kinase inhibitors, sirtuin inhibitors and low doses of actinomycin D will be described among other examples. The possibility of using combinations of molecules will also be analyzed as will the issues of toxicity and resistance expected from these types of mol- ecules. One concern is that the activation of p53 might exert some specific harmful affect on normal tissues and another is that treat- ment with p53 activators will select for tumor cells in the popula- tion that have mutant p53. What can be done in those cases where p53 is mutant? One approach is the search for molecules that re-activate mutant p53 proteins, though some have likened this to trying to unscramble eggs recent discoveries in protein dynamic and protein folding pathways have been very exciting. Recent approaches to developing molecules that will chaperone mutant p53’s to fold in the active state will be described with ref- erence to recent crystallographic analysis. A second conceptual approach to exploiting the frequent loss of p53 function in tumors is that of synthetic lethality. Might p53 mutant tumors be more susceptible to inhibitors of DNA repair as has been seen for BRCA1 mutant tumors for example? Finally the concept of using p53 activating molecules as chemo-protectives has been proposed. In this concept normal cells are arrested temporarily in GI by p53 activators such as the Mdm2 inhibitor Nutlin. These cells are thus protected from cytotoxic drugs active at G2/M such as taxol. Cells that lack p53 and are not therefore arrested by Nutlin would retain their sensitivity. In this model then the therapeutic index of such cytotoxic drugs for p53 mutant tumors would be enhanced by protection of normal tissues. References: Klein C and Vassilev LT. B J Cancer 2004; 91:1415 Dey A, Verma CS and Lane DP. B J Cancer 2008; 98:4 IUBMB LECTURE PL 2 Genomes, structural biology and drug discovery: challenges for academia and industry T. Blundell Biochemistry, Cambridge University, Cambridge, UK Over the past two decades funding for research and development in drug discovery in the pharmaceutical industry has increased exponentially, but the returns in new drug approvals on the mar- ket have not been commensurate with this investment. What can we as cell, molecular and structural biologists do to make the early stages of drug discovery more effective and efficient? The knowledge that is now emerging from genomics of man and pathogens and from targeted structural proteomics programmes has the potential to accelerate drug discovery. DNA and protein sequences, in particular non-synonymous single nucleotide poly- morphisms and somatic mutations, when taken together with structural and functional information on the gene products, can provide insights into the relationship of human genetic variation and disease. This is also helpful in identifying new targets for drug discovery; it is an exploration of biological space. On the other hand, high-throughput biophysical and structural analyses can be used to investigate the chemical molecules that proteins might bind; this is an exploration of chemical space. I will argue that this is best achieved by fragment-screening techniques, which inform not only lead discovery but also optimization of candi- date drug molecules. I will describe such developments not only in industry for diseases that are major challenges in the West but also in academia for diseases of poverty, rare diseases and diffi- cult targets. A long-term objective must be to define the chemical space around all macromolecules in man and in pathogens, so as not only to facilitate lead discovery but also to identify potential off-target interactions and minimise side effects. WOMEN IN SCIENCE LECTURE PL 3 How myosin motors work: a fun journey from structure to function A. Houdusse Section de recherche, Institut Curie, Paris cedex 05, FRANCE Directed movement is essential for life. To decipher the mecha- nisms that allow a cell to migrate, to divide and organise the intra-cellular traffic between various cellular compartments, stud- ies of the molecular motors involved in these functions is critical. Every cell in our bodies contains about 100 different proteins that act like molecular motors in carrying out various forms of movement. To understand how a molecular motor functions, sev- eral sophisticated approaches have all made their contribution. In particular, one family of motors, the actin-based myosin motor is now one of the best-understood enzyme thanks to the insights from molecular genetics, biochemical, biophysical and structural studies, including single molecule physical approaches to measure force and step size. Myosins are critical for many of these movements – from muscle contraction to cytokinesis, intra- cellular traffic and sophisticated cellular functions such as hear- ing. Deficit in these motors can lead to a number of human genetic disorders. Force is produced by these molecular motors by converting chemical energy derived from ATP hydrolysis into mechanical energy via the interaction with their track, the actin filament. A number of approaches have provided detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes in the motor domain are coupled by a lever-arm mecha- nism to a working stroke of several nanometers. Structural stud- ies coupled with functional studies is a very powerful technique to identify at atomic resolution a number of communication pathways that are involved at different steps of the ATPase cycle to couple the nucleotide-binding pocket with the actin interface and the lever arm movement. A reverse motor, myosin VI has Plenary Lectures Abstracts FEBS Journal 276 (Suppl. 1) 1–3 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 1 been particularly intriguing and informative because of its atypi- cal motility properties. The current questions regarding the details of force production and the current challenges to under- stand how these motors are regulated, recruited and how the spe- cific cellular functions of several motors are coordinated to accomplish a particular function in the cell will also be reviewed. SIR HANS KREBS LECTURE PL 4 Membrane rafts: important (yet controversial) components of receptor signaling V. Horejsi Institute of Molecular Genetics AS CR, Prague 4, CZECH REPUBLIC Membrane rafts (microdomains enriched in glycosphingolipids, cholesterol, glycosylphosphatidylinositol-anchored proteins, Src- family kinases and G-proteins, markedly resistant to solubiliza- tion in certain detergents) appear to play important roles in early phases of immunoreceptor signaling, but also in regulation of a number of other receptors. Most transmembrane proteins are excluded from these specialized areas of membranes, notable exceptions being several palmitoylated proteins such as the T- lymphocyte co-receptors CD4 and CD8, and several recently described transmembrane adaptor proteins (TRAPs) such as LAT, NTAL/LAB, PAG/Cbp and LIME. All these molecules possess a short N-terminal extracellular peptide, transmembrane segment followed by a palmitoylation motif (CxxC) and cytoplas- mic domain containing tyrosine motifs potentially phosphory- lated by the Src- or Syk-family kinases. Tyrosine-phosphorylated transmembrane adaptors bind other signaling molecules (several cytoplasmic adaptors and enzymes) and thus organize ‘signalo- somes’. The very existence of lipid rafts in vivo has been repeat- edly questioned, as they are difficult to visualize by current microscopic techniques and most information on them comes from biochemical studies in detergent lysates. Little is known about properties of these controversial membrane complexes in vivo, their possible heterogeneity and its relation to their signaling functions. Recent studies from our and other laboratories have identified a new type of non-conventional ‘heavy rafts’ that are able to support at least partially T-cell receptor signaling. These ‘heavy rafts’ are apparently distinguished from the the ‘classical rafts’ by higher protein/lipid ratio and are, similarly to the ‘classi- cal rafts’, resistant to mild detergents (Brij-98, -58), sensitive to other mild detergents (laurylmaltoside, octylglucoside) and con- tain a number of functionally important transmembrane proteins. The novel type of membrane rafts can also support, albeit less effectively, immunoreceptor signaling. THE EMBO LECTURE PL 5 Molecular machines driving plant growth D. Inze, N. Gonzalez and G. De Jaeger UGent-VIB Research, Department of Plant Systems Biology, Gent, BELGIUM Understanding the mechanisms that control tissue, organ and organism size are amongst the most mysterious and fascinating open questions in biology. A key concept in ‘size biology’ is that both, in animals and plants, size itself is regulated. Our long term goal is to unravel the molecular pathways that govern leaf size in Arabidopsis. Our approach is based on studying the action mech- anisms of genes which, when mutated or overexpressed, enlarges leaf size [hereafter called ‘intrinsic yield genes’ (IYG)] (Gonzalez et al., 2009). Currently, we have confirmed the positive effect of 13 IYGs on Arabidopsis leaf size. In all cases examined so far enlarged leaf size results from an increased cell number without affecting significantly cell size, pinpointing to a central role of cell proliferation in size control. To understand how the cell cycle is linked to size, tandem affinity purification of > 100 proteins involved in cell cycle as well as size control (IYGs) were carried out. Transcript and metabolome profiling has been undertaken on various IYG lines. Detailed computational as well as func- tional analysis has shed new light on how organ size is governed in plants. Reference: Gonzalez N, Beemster GTS and Inze ´ D (2009). David and Goli- ath: What can the tiny weed Arabidopsis teach us to improve biomass production in crops? Curr. Opin. Plant Biol. In press THEODOR BU ¨ CHER LECTURE PL 6 The molecular basis of cytokine receptor signalling – theme and variations W. Sebald Theodor-Boveri-Institut fu ¨ r Biowissenschaften, Biozentrum, Wuerzburg, GERMANY Cytokine receptors are crucial for the maintenance, regulation and growth of cells in multicellular organisms. As a common theme in cytokine signalling single-span receptor chains are assembled in the cell membrane by a ligand enabling cross-activa- tion of the aligned cytoplasmic receptor domains. Nature has cre- ated many variations of how this general principle is realized in a cell. This lecture will focus on cytokines of the 4-helix bundle (In- terleukins) and cystine-knot (TGF-b/BMP) families. Receptor chains upon activation can form duos, trios, quartets, and even larger assemblies. Homo- and heteromeric complexes exist. Chains function as triggers (initiating the cytosolic signal), drivers (transactivating other chains), or affinity regulators (increasing the potency of the ligand). Common chains are used by several cytokine families. The structure of the extracellular ligand-bind- ing domain (ECD) of a number of these receptor complexes have now been elucidated providing the molecular basis for under- standing the functional relevance of this mechanistic diversity in a cellular context. Biochemical and structural data have revealed ligand recognition mechanisms. Contact sites are usually large and rather flat. A limited number of contact residues provide most part of the binding free energy (hot spots). Leaks in hydro- phobic seals appear to provide a mechanism for adjusting the affinity of a hot-spot interaction (scalability). Promiscuous inter- action with common chains under retention of specificity is typi- cally associated with hydrophobic interfaces containing multiple water molecules. In particular BMP ligands are promiscuous and interact not only with receptors but also with a multitude of modulator proteins which inhibit or enhance BMP signalling. Receptor complexes often comprise high- and low-affinity chains. Low affinity interactions are likely reinforced by co-localization of the binding partners in the membrane. Cytokine receptor sys- tems offer promising targets for drug development. Information on structure and activation mechanism provides leads for devel- oping biologicals, as (1) engineered cytokines, (2) cytokine mutants acting as receptor antagonists, (3) receptor ECD-Fc fusion proteins and (4) mAbs acting as ligand traps and inhibi- tors of cytokine signalling. Possible indications exist in the areas of haematology, immunology, inflammation, cancer, and tissue regeneration. Abstracts Plenary Lectures 2 FEBS Journal 276 (Suppl. 1) 1–3 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies PABMB LECTURE PL 7 Translational control via mRNA 3’–5’ interactions: from development to miRNA function N. Sonenberg Department of Biochemistry, McGill University, Montreal, CANADA Translational control, which is mediated by mRNA 3’–5’ interac- tions, is widespread. The eukaryotic mRNA 3’ poly (A) tail acts synergistically with the 5’ cap structure to enhance translation. This effect is mediated by a bridging complex, composed of the poly (A) binding protein (PABP), eIF4G, and the cap-binding protein, eIF4E, which brings about mRNA circularization. The bridging between the mRNA 3’ and 5’ ends is controlled by sev- eral proteins that bind to either eIF4E or PABP and regulate translation initiation. Translation initiation is also controlled by proteins, which bind simultaneously to eIF4E and the 3’ UTR of the mRNA. These proteins function, for example, during devel- opment by inhibiting translation of specific mRNAs in a tempo- ral and spatial manner. The Drosophila Bicoid protein inhibits translation of caudal mRNA in the anterior of the embryo, by interacting simultaneously with the caudal 3’ UTR and an eIF4E cognate protein, 4EHP. MicroRNAs (miRNAs) comprise a large family of small ~21-nucleotide-long non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans and plants. In mammals, miRNAs are predicted to control the activity of ~50% of all protein-coding genes and have been shown to participate in the regulation of almost every cellu- lar process investigated to date. The post-transcriptional control of gene expression by miRNAs is mediated by RISC (RNA- induced silencing complex), which binds mostly to the mRNA 3’ UTR. We have developed a mammalian cell-free extract from mouse Krebs-2 ascites cells that recapitulates the miRNA response in cells. Using this system, we demonstrated that endog- enous let-7 miRNA rapidly inhibits cap-dependent translation initiation of miRNA-targeted mRNA reporters. Addition of the cap-binding protein complex, eIF4F, alleviated translational repression, suggesting that miRNAs function at least in part by antagonizing the cap recognition process. miRNAs also direct deadenylation of miRNA-targeted mRNAs through the activity of RISC. The deadenylation occurs after the initial inhibition of translation initiation. Thus, miRNAs inhibit translation by affecting the mRNA 3’ and 5’ ends. FEBS JOURNAL PRIZE LECTURE PL 8 The Janus-faced atracotoxins are specific blockers of invertebrate K Ca channels S. J. Gunning 1 , F. Maggio 2 , M. J. Windley 1 , S. M. Valenzuela 1 , G. F. King 3 and G. M. Nicholson 1 1 Neurotoxin Research Group, Department of Medical Molecular Biosciences, University of Technology, Sydney, AUSTRALIA, 2 Department of Molecular, Microbial Structural Biology, Univer- sity of Connecticut School of Medicine, Farmington, CT, USA, 3 Division of Chemical and Structural Biology, Institute for Molecu- lar Bioscience, University of Queensland, Brisbane, AUSTRALIA The j-atracotoxins (j-ACTXs: formerly Janus-faced atracotox- ins) are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selec- tive, high-affinity blockers of invertebrate Ca 2+ -activated K + (K Ca ) channels. j-ACTX-Hv1c, the prototypic member of this toxin family, selectively blocked K Ca channels in cockroach unpaired dorsal median neurons with an IC 50 of 2 nM, but it did not significantly affect a wide range of other voltage-activated Ca 2+ or Na + channel subtypes. j-ACTX-Hv1c blocked heterolo- gously expressed cockroach large-conductance Ca 2+ -activated K + (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-depen- dent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of j-ACTX-Hv1c was established by its failure to sig- nificantly inhibit mouse mSlo channels (IC 50 >10lM) and its lack of activity on rat dorsal root ganglion neuron K Ca channel currents. This study establishes the j-atracotoxins as valuable tools for the study of invertebrate K Ca channels and suggests that K Ca channels might be potential insecticide targets. FEBS LETTERS YOUNG SCIENTIST LECTURE PL 9 Chaperone-signal peptide interactions on the Tat protein transport pathway S. J. Coulthurst and F. Sargent Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, Scotland, UK The twin-arginine transport (Tat) pathway is a protein targeting system dedicated to the transmembrane translocation of fully folded proteins. This system is highly prevalent in the cytoplas- mic membranes of bacteria and archaea, and is also found in the thylakoid membranes of plant chloroplasts. Proteins are targeted to a membrane-embedded Tat translocase by specialised N-term- inal twin-arginine signal peptides bearing the distinctive SRRxFLK ‘twin-arginine’ amino acid motif. The Escherichia coli system is one of the best characterised and here the Tat translo- case contains a ‘signal recognition’ module, comprising the TatBC proteins, and a ‘transport channel module’ comprising multiple copies of TatA. These two modules work together by a largely unknown mechanism to couple the proton motiveforce directly to protein transport. Escherichia coli produces 27 Tat-tar- geted proteins and the majority of these are cofactor-containing enzymes that must acquire their prosthetic groups, and some- times associate with partner subunits, in the cytoplasm before export. The processes that govern cofactor-loading and protein folding, and co-ordinate these events with protein export, are beginning to be unearthed. One of the best-studied E. coli Tat substrates is the trimethylamine N-oxide reductase (TorA), which is a molybdopterin-containing enzyme. Assembly and export of TorA is regulated by TorD, which performs a quality control task known as ‘Tat proofreading’ in order to prevent premature targeting of TorA until all biosynthetic processes are complete. Tat proofreading involves the direct physical interaction between TorD and the TorA signal peptide. The molecular basis of the signal peptide recognition and release mechanism is currently under investigation. Plenary Lectures Abstracts FEBS Journal 276 (Suppl. 1) 1–3 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies 3 . PLENARY LECTURES DATTA LECTURE PL 1 Drug discovery in the p53 pathway D. Lane Division. actin interface and the lever arm movement. A reverse motor, myosin VI has Plenary Lectures Abstracts FEBS Journal 276 (Suppl. 1) 1–3 (2009) ª 2009 The Authors