Báo cáo khoa học: PLENARY LECTURES pdf

The emerging understanding of telomeres and telomerase, in both normal and transformed cells, holds promise for improv-ing health and combattimprov-ing cancer.. Grummt German Cancer Rese

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 dividcaus-ing 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 combattimprov-ing 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 settinclud-ing 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-cancan-cer 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 phenogeno-types 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

mechanisms for allowing genetic diversity and fluctuating

environ-ments to fuel the pace of evolutionary change

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 generates1O2upon blue light illumination.1O2 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

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 methylapropor-tion 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 1a and decreased mtDNA and PGC-1a mRNA We provide evidence for PGC-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 Munkonda1, M Fausther1, V V Ivanenkov2, J Pelletier1,

A Tremblay1, B Ku¨nzli3, T L Kirley2and J Se´vigny1

1

Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Que´bec, Universite´ Laval, Que´bec,

QC, Canada,2Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA,3Department of General Surgery, Universita¨t Mu¨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

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-splicrepeat-ing 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