Báo cáo khoa học: Symposia Lectures pdf

LcrV forms a structure at the tip of the needle and this structure is believed to act as a scaffold for the insertion of a pore made of YopB and YopD into the target cell membrane.. Stru

Monday, July 9, 2007

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J Diffley

No abstract available

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Molecular bioimaging of gene transcription

P Cramer

Gene Center, Munich, GERMANY

In the first half of the talk, the current state of our ongoing structure-function analysis of the mRNA transcription cycle will be dis-cussed The crystallographic structures of the complete 12-subunit RNA polymerase II in free form and with bound DNA and RNA have been determined as atomic models The structure of the complete Pol II elongation complex bound by the transcript cleavage factor TFIIS explained how Pol II uses a single tunable active site for both RNA synthesis and RNA cleavage Three structures of complexes

of CTD-interacting proteins provided insights into the coupling of transcription elongation to mRNA processing and histone methyla-tion, and into Pol II recycling The first structure of a Pol II complex with an RNA inhibitor and the first atomic structures of subcom-plexes of the mediator of transcriptional regulation provided insights into gene regulation In the second half of the talk, recently published and unpublished data will be presented that will show the directions for future investigations A set of new structures of Pol II bound to damaged DNA together with complementary functional assays revealed an unexpected mechanism of lesion recognition during transcription-coupled DNA repair Soon we will obtain new insights into the structure and function of the distantly related RNA polym-erase I, which produces ribosomal RNA This part of the talk will highlight the complementary use of various structural (X-ray, EM, NMR) techniques and functional studies in vitro and in vivo

18

Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells

Y Watanabe

University of Tokyo, Tokyo, JAPAN

Centromeric protection of cohesin from separase-cleavage at meiosis I is a hallmark of reductional chromosome segregation, an essential step to reduce ploidy, and potentially related to human birth defects We here demonstrate that mammalian shugoshin protein Sgo2 is responsible for this protection in oocytes However, Sgo2 also protects cohesin in mitosis from the so-called prophase pathway, but never at the onset of anaphase of mitosis as well as meiosis-II in spite of its localization at centromeres We found that Sgo2 colocalizes with cohesin at the inner centromere in mitotic prophase and meiosis-I (where centromeres are mono-oriented and not under tension), but they come apart during mitosis and meiosis-II, depending on tension across bi-oriented centromeres Accordingly, we demonstrate that the tension on mitotic chromosomes displaces shugoshin within centromeres and de-protects cohesin, since cohesin can be dissoci-ated from centromeres at metaphase arrest even prior to onset of anaphase Our study thus solves the puzzle of why the same shugoshin molecule can protect cohesin at anaphase onset of meiosis-I but neither of meiosis-II nor mitosis, providing insight into the evolution of centromeric protection for meiotic chromosome segregation

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RNAi and gene silencing in heterochromatin

D Moazed

Harvard Medical School, BOSTON, MA, USA

Assembly of heterochromatin at the fission yeast centromeres requires components of the RNAi machinery as well as histone binding proteins and modifying enzymes We have identified several multiprotein complexes that link the RNAi pathway to heterochromatin assembly, and have analyzed their activities in vitro and in vivo Our results suggest that RNAi complexes associate with nascent RNA transcripts to initiate and maintain gene silencing The mechanism of silencing involves the co- transcriptional degradation of RNAs that are transcribed in heterochromatic domains We refer to this novel silencing mechanism as CTGS (Co-Transcriptional Gene Silencing)

M Matzke

GMI, Vienna, AUSTRIA

No abstract available

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Assembly and operation of the Yersinia injectisome

G R Cornelis

Biozentrum, Basel, SWITZERLAND

The type III secretion injectisome is a nanosyringe that injects bacterial effector proteins into the cytosol of eukaryotic cells It is related

to the flagellum, with which it shares structural and functional similarities It consists of a basal body made of several rings spanning the bacterial membranes, connected by a central tube On top of the basal body, comes a short stiff needle The basal body contains the export apparatus, which serves first for the export of the needle subunits and later for effectors This structure is sufficient for exporting effector proteins across the two bacterial membranes but not to inject them into the cytosol of target cells This translocation step requires three more proteins, LcrV, YopB and YopD, in the case of Yersinia LcrV forms a structure at the tip of the needle and this structure is believed to act as a scaffold for the insertion of a pore made of YopB and YopD into the target cell membrane LcrV is known since the mid nineteen fifties to be a protective antigen against plague The length of the needle is controlled by a mechanism involving a protein thought to act both as a molecular ruler and a substrate specificity switch When assembly of the needle is complete, the molecular ruler changes the substrate specificity of the export apparatus, which becomes ready to export pore formers and the effec-tors Export of the latter will only occur upon contact with a target cell

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Structure, assembly and function of the Salmonella type III protein secretion nanomachine

J E Galan

Yale University School of Medicine, New Haven, CT, USA

A central component of type III secretion systems is a multi-protein organelle known as the needle complex This organelle is composed

of a multi-ring based anchored to the bacterial envelope, an inner rod embedded within the base, and a slender extension or needle that protrudes from the bacterial surface Assembly of the needle complex occurs through a series of discrete and well-coordinated steps resulting the assembly of the base substructure This substructure operates as a ‘type III secretion machine’ but exclusively dedicated to the export of the components of the inner rod and needle substructures Upon completion of the assembly of the entire needle complex, the type III secretion machine switches substrate specificity becoming competent for the secretion of effector proteins The details of the type III secretion organelle assembly process and its function will be discussed

References

1 Kubori T, Matsushima Y, Nakamura D, Uralil J, Lara-Tejero M, Sukhan A, Galan JE, Aizawa S.-I Science 1998; 280: 602–605

2 Kubori TK, Sukhan A, Aizawa S.-I Galan JE Proc Natl Ac Sci USA 2000; 97: 10225–10230

3 Marlovitz TC, Kubori T, Sukhan A, Thomas DR, Galan JE, Unger UM Science 2004; 306: 1040–1042

4 Akeda Y, Galan JE Nature 2005; 437: 911–5

5 Marlovitz TC, Kubori, T., Lara-Tejero, M., Thomas, D R., Unger, V M., and J E Galan 2006 Nature 441(7093):637–40

6 Galan JE, Wolf-Watz H Nature 2006; 444(7119): 567–73

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mRNA localization and polarized growth in yeast

J E Gerst, R Gelin-Licht, L Haim, L Yakir-Tamang and G Zipor

Weizmann Institute of Science, Rehovot, ISRAEL

Targeted mRNA trafficking may play a pivotal role in determining the local concentration of protein and, thus, influence a number of cellular processes, such as polarization, division, and body morphogenesis By using the yeast Saccharomyces cerevisiae, we demonstrated that mRNAs encoding polarity and secretion factors (POLs; i.e Sro7, Cdc42, Sec4) involved in polarity establishment and polarized growth are trafficked to the presumptive bud site and localize to the bud tip during division As POL mRNA trafficking precedes POL protein enrichment and budding, it suggests that mRNA transport facilitates polarized growth Furthermore, asymmetric mRNA traf-ficking is directly connected with the transport of cortical ER (Aronov et al Mol Cell Biol 2007; 27: 3441–55) These axioms also hold true for haploid cells treated with mating factor, which develop polarized membrane extensions (shmoos) analogous to neural or podial projections in higher organisms However, in contrast to budding yeast, two mRNA trafficking mechanisms – one SHE-dependent and the other SHE-independent – operate during shmooing Other work demonstrates that non-polarized mRNAs, such as those encoding peroxisomal and mitochondrial proteins, are also targeted to specific subcellular destinations Thus, all mRNAs have a specific pattern

of distribution and we are developing novel techniques to localize endogenous mRNAs in vivo (Haim et al Nat 2007 Methods, pub online)

Genome-wide quantitative image analysis of endocytosis and signalling

C Collinet, Y Kalaidzidis and M Zerial

Max Planck Institute of Molecular Cell Biology and Genetics MPI-CBG, Dresden, GERMANY

Endocytosis is an essential process serving multiple key cellular functions, such as nutrient uptake, signal transduction, and defence against pathogens Small GTPases of the Rab family act as membrane organizers, specifying the structural and functional identity of endosomes Various Rab GTPases occupy distinct membrane domains on early endosomes (Rab5, Rab4 and Rab11) or late endosomes (Rab7 and Rab9), termed ‘Rab-domains’ Rab-domains are dynamically assembled and disassembled on the membrane, as in the case of the Rab5-domain which is disassembled and replaced with Rab7 in the course of transport from early to late endosomes To identify other components of the endocytic apparatus we performed high-content image-based genome-wide RNAi screening in human cells The genes were identified on the basis of a multi-parametric analysis comprising ~40 parameters such as endosome size, intracellular distribu-tion and concentradistribu-tions of labeled proteins, and a set of statistical procedures Selected genes were subjected to an in-depth analysis, using fast live cell imaging and novel image analysis algorithms, including particle detection and tracking algorithms This multi-para-metric analytical platform is used to develop a mathematical model that can quantitatively predict endosome dynamics and signalling properties under various physiological and pathological conditions

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Alternative paths to lysosomes

H Stenmark

The Norwegian Radium Hospital, Oslo, NORWAY

Proteasomes and lysosomes are the major sites for protein degradation in the cell Whereas lysine-48-linked polyubiquitin chains target cytosolic substrate proteins for proteasomal degradation, monoubiquitin, often attached at multiple target lysines, is frequently used as a signal for lysosomal degradation of membrane proteins A special case of lysosomal protein degradation involves autophagy, the seques-tration of portions of cytoplasm by a double-membraned structure called the autophagosome When the autophagosome fuses with a lysosome, the sequestered material will be degraded We have been studying lysosomal protein transport in two contexts: Lysosomal trafficking of endocytosed growth factor receptors as a mechanism of ligand-mediated receptor downregulation, and autophagy of cyto-plasmic positive protein aggregates as a mechanism of neuroprotection Central in these processes are complexes of ubiquitin-binding proteins that mediate lysosomal sorting We are now elucidating the structures and functions of these sorting complexes

22

Road to ruin: apoptotic pathways in the nematode C elegans

M Hengartner

University of Zu¨rich, Zu¨rich, SWITZERLAND

No abstract available

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Protease signalling: cysteine cathepsins pathways to cell death

B Turk

J Stefan Institute, Ljubljana, SLOVENIA

Apoptosis is the major way of eliminating potentially harmfull and superfluous cells The pathway is severely impaired in cancer and cancer cells generally fail to die Proteolysis plays an essential role in the process with caspases being the major players However, there

is increasing evidence that lysosomal proteases are actively involved in apoptosis Among them the most abundant and best characterized are cysteine cathepsins, including cathepsins B and L, and the aspartic protease cathepsin D A critical point is lysosomal membrane per-meabilization (LMP), which leads to translocation of lysosomal proteases to cytosol Despite considerable evidence, the signaling path-ways downstream of LMP are not well understood In order to study these mechanisms, we have been using a model system using the lysosomotropic compound LeuLeu-OMe, which was shown to significantly reduce the graft-versus-host disease in a mouse model We were able to show in eight cellular models that selective lysosome disruption with Leu-Leu-OMe resulted in apoptosis, characterized by lysosome disruption, translocation of lysosomal proteases to the cytosol and subsequent activation of caspases indirectly through mito-chondria disruption The proapoptotic Bcl-2 family member Bid was found to be a cellular target for cysteine cathepsins in most cells used In addition to Bid, some other cathepsin cellular targets were also identified and will be further discussed, as well as the pathways

in the absence of caspase activity

Functional characterization of the cylindromatosis tumor suppressor

G Mosialos1,2, E Trompouki2, A Tsagaratou2and D Kontoyiannis2

1Aristotle University of Thessaloniki, Thessaloniki, GREECE,2BSRC Al Fleming, Vari, GREECE

The cylindromatosis tumor suppressor gene (cyld) has been associated with the development of various pathologies Mutations in cyld predispose humans to the development of tumors of skin appendages and cyld downregulation has been linked to inflammatory disor-ders and cancer of the colon and liver Cyld encodes a 956 amino acid cytoplasmic protein (CYLD) which has deubiquitinating activity that targets preferentially lysine-63-linked polyubiquitin chains CYLD is a negative regulator of NF-kappaB and JNK activation by members of the tumor necrosis factor receptor superfamily The deubiquitinating activity of CYLD has been linked genetically to its NF-kappaB inhibitory and tumor suppressing activities In order to characterize the function of CYLD in vivo we have generated mice with targeted inactivation of the catalytic domain of CYLD in a conditional format Interestingly, mice with inactivated CYLD in all tis-sues (cyld–/–) died shortly after birth, from apparent respiratory failure No major developmental defects were noted Histological exam-ination of lung tissue from E18.5 embryos revealed a higher overall density with abnormal alveolar epithelia and septa in samples from cyld–/– embryos compared to samples from wild type embryos Tissue specific inactivation of CYLD in thymocytes and macrophages revealed an essential role for CYLD in thymocyte positive selection and macrophage response to inflammatory stimuli Our experiments have revealed a pleiotropic function of CYLD in mammalian physiology The molecular mechanisms that underlie the phenotypic char-acteristics of CYLD inactivation will be discussed

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Genetic analysis of the mammalian cell cycle: Cdk1 is sufficient to drive mammalian cell division

M Barbacid

Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid, SPAIN

We and others have shown that none of the four kinases implicated in driving interphase during the mammalian cell cycle, Cdk2, Cdk3, Cdk4 and Cdk6, are essential for cell division Likewise, we have reported that most cells proliferate well in the absence of Cdk4 and Cdk6, Cdk6 and Cdk2 (Malumbres et al., Cell 2004; 118: 493) and more recently, in the absence of Cdk4 and Cdk2 (Barrier et al Mol Oncology, in press) We have now generated embryos lacking all interphase Cdks Triple knock out Cdk4(-/-);Cdk6(-/-);Cdk2(-/-) embryos (Cdk3 is not functional due to a natural mutation) develop until midgestation and display normal levels of cell proliferation and organogenesis As expected, they do not survive to term due to lack of proliferation of hematopoietic cells, a defect attributed to the absence of Cdk4 and Cdk6 This defect appears to be somewhat exacerbated by the absence of Cdk2 Cdk4(-/-); Cdk6(-/-); Cdk2(-/-) MEFs proliferate in culture and become immortal upon serial passage However, these cells display an extended interphase and prolifer-ate more slowly than wild type MEFs or MEFs expressing just one interphase Cdks pRb is phosphorylprolifer-ated at well known sites in these triple knock out cells Thus, mammalian cells can divide with just Cdk1 Ablation of Cdk1, however, prevents cell division These obser-vations, taken together, indicate that ‘interphase Cdks’ are required to ensure proliferation of specific cell types but are not essential to drive the basic mammalian cell cycle as previously thought

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Distinct mechanisms of tumor invasion and metastasis

G Christofori

Institute of Biochemistry and Genetics, Basel, SWITZERLAND

Tumor cell invasion into the surrounding tissue can exhibit a phenotype of either single cell or collective cell migration Single cell migra-tion is mainly dependent on signaling pathways within migrating cells themselves and is usually accompanied by an epithelial-mesenchy-mal transition (EMT), which involves several genetic and epigenetic alterations including the loss of E-cadherin and the gain of N-cadherin expression (the cadherin switch) Gene expression profiling experiments together with biochemical analysis reveal that the dis-tinct stages of EMT are tightly regulated by epistatic cascades of transcriptional control circuits which involve activation and repression

of a large number of genes modulating the migratory and invasive behavior of tumor cells In contrast, collective cell migration requires the maintenance of cell-cell adhesion and a certain multicellular organization of the tumor tissue Recently, we have shown that the expression of podoplanin, a small mucin-like protein, is upregulated in a number of human carcinomas, in particular squamous cell car-cinomas Podoplanin induces tumor cell spreading, migration and invasion in vitro and in vivo by a novel molecular pathway of collec-tive invasion that does not involve the loss of E-cadherin function or EMT.Finally, we propose a third type of metastatic tumor cell dissemination which involves an upregulated expression of lymphangiogenic factors, such as vascular endothelial growth factors C and

D, and the subsequent induction of lymphangiogenesis Increased lymphatic vessel density within and around expanding tumors leads to lymph node metastasis, most likely by a passive washing out and trapping of tumor cell clusters in regional lymph nodes

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The crucial role of mitochondria in iron-sulfur protein biogenesis and cellular iron metabolism

R Lill

Zytobiologie, Marburg, GERMANY

Research over the past few years has established the crucial role of mitochondria in both the biogenesis of iron-sulfur (Fe/S) proteins and the regulation of cellular iron homeostasis The two processes are coupled mechanistically in that defects in Fe/S protein biogenesis elicit increases in both cellular and mitochondrial iron import The molecular basis of Fe/S cluster synthesis and assembly into apopro-teins in a living cell has been subject to intense research activities (see Review) Biogenesis is accomplished by three complex proteina-ceous machineries Mitochondrial Fe/S proteins require the iron-sulfur cluster (ISC) assembly machinery which was inherited from bacteria during evolution Cytosolic/nuclear Fe/S protein assembly also requires the function of this machinery, yet additionally depends

on the mitochondrial ISC export apparatus and the cytosolic iron-sulfur protein assembly (CIA) machinery The components of all three systems are highly conserved in eukaryotes from yeast to man suggesting similar mechanisms of Fe/S protein assembly We are currently most interested in defining the molecular basis of the contribution of mitochondria for cytosolic/nuclear Fe/S protein biogenesis The process is of importance for human disease Depletion of frataxin leads to the neurodegenerative disease Friedreich’s ataxia, and a defect

in ABCB7 is associated with X-linked sideroblastic anemia and ataxia (XLSA/A)

Reference

1 Lill R, Mu¨hlenhoff U Iron-sulfur protein biogenesis in eukaryotes: components and mechanisms Annu Rev Cell Dev Biol 22, 457–486

Cytochrome c binding to the cytochrome bc1 complex: an interaction critical for electron transfer

S Solmaz, T Wenz, U Scha¨fer, A Nyola and C Hunte

Max-Planck-Institute of Biophysics, Frankfurt am Main, GERMANY

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins Structural information about specific and transient complex formation is scarce In cellular respiration, the mobile electron carrier cytochrome c shuttles electrons from the cytochrome bc1 complex to cytochrome c oxidase The x-ray structure of the complex between cytochrome c and cytochrome bc1 complex at 2.97-A˚ resolution gave first structural insight for such a complex from the respiratory chain [Lange and Hunte, Proc Natl Acad Sci USA 2002] The structure revealed the nature of the interface, which is clearly well suited for transient interaction and fast turnover The close spatial arrangement of the cofactors suggests a direct heme-to-heme electron trans-fer Remarkably, cytochrome c binds to only one recognition site of the homodimeric complex We now determined the structure of the electron transfer complex in the reduced state at 1.9 A˚ resolution [Solmaz and Hunte, in prep] The high resolution allows an accurate description of the interface, focussing especially on the role of the electrostatic and water-mediated interactions Comparison with a sec-ond structure obtained for the isoform-2 of cytochrome c bound to the cytochrome bc1 complex lead to the definition of a minimal interface, the core interface, which is present in all of these structures The importance of single residues of the core interface has been challenged by site-directed mutagenesis and characterization of the variants in respect to complex formation and electron transfer This work is also of general interest for the area of weakly associated protein complexes

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Dynamics of the photosynthetic membrane during acclimation to a changing light environment

J Rochaix

University of Geneva, Geneva, SWITZERLAND

Photosynthetic organisms are able to adjust to changes in light quality and to maintain a high photosynthetic yield through state transi-tions This process leads to a balancing of the light excitation energy between the light harvesting systems (LHC) of photosystem II (PSII) and photosystem I (PSI) Preferential stimulation of PSII relative to PSI leads to the reduction of the plastoquinone pool and to the activation of a protein kinase which phosphorylates LHCII, a process which induces the displacement of LHCII from PSII to PSI (state 2) Excess stimulation of PSI leads to the reverse process (state 1) We have used a genetic approach in Chlamydomonas with the ultimate aim of identifying some of the key components involved in the signaling chain of state transitions We have identified a chloro-plast thylakoid-associated Ser-Thr protein kinase Stt7 which is required for the phosphorylation of LHCII and for state transitions Stt7 interacts with LHCII, the cytochrome b6f complex and PSI, but not with PSII Stt7 is related to the Stl1 kinase of Chlamydomonas and

to the protein kinases STN7 and STN8 of Arabidopsis STN7 is also required for LHCII phosphorylation and state transitions and there

is some synergy between these two kinases Comparison of this acclimation process in a green motile unicellular alga and a sessile land plant has provided interesting insights into evolutionary aspects of this response

26

Learning and innovation of multi-enzyme complexes: cooperation in a network context

P Csermely

Semmelweis University, Budapest, HUNGARY

The network and game theory paradigms are increasingly used to describe the topology and dynamics of complex systems Protein and multi-enzyme complexes can be perceived as parts of large protein-protein interaction and metabolic networks with overlapping modules These networks are small-worlds, have a non-negligible amount of hubs, and contain long-range, low affinity contacts providing network integrity and stability Elements of these networks are in a dynamic contact with each other Our recent results show that learning and innovation are both needed to keep a satisfying level of cooperation in continuously changing, dynamic networks The lecture will imply these and other recent results of network topology and dynamics to the topological analysis of protein structures, protein complexes and multi-enzyme complexes As a part of this, we will show the role of hubs and central network elements in governing enzyme activity, al-losteric regulation, protein motor function, signal transduction and protein stability The importance of central network elements enriched in active centers and ligand binding sites directing the dynamics of the entire protein will also be discussed Stepping a level higher, we will assess cooperation between members of protein and multi-enzyme complexes Changes in network formation during stress and ageing will also be introduced Finally, we will discuss possible network-based strategies to expand the number of drug targets and

to overcome the current ‘target-shortage’ by introducing system-designed multi-target drugs

D2-L5

Multistep catalysis by nanomachines

R Perham

University of Cambridge, Cambridge, UK

Many biochemical transformations are multistep reactions and incapable of being catalysed by a single enzyme active site Thus many multistep transformations are catalysed by large assemblies of enzymes, which may have beautiful and elaborate quaternary structures that contribute to the catalytic mechanism In some instances, physical tunnels connect the active sites and allow intermediates to diffuse between them; in other instances, prosthetic groups in the form of covalently attached swinging arms, such as lipoyl groups, biotinyl groups and phosphopantetheine groups, serve to transfer the catalytic intermediates These unusual structural features introduce aspects

of enzyme catalysis that go beyond what can be expected of individual uncomplexed enzymes; among them we can identify enhancement

of catalytic activity brought about by the retention of intermediates at high local concentrations, substrate channeling to negate compet-ing side reactions, and the protection of catalytic intermediates from the degradation they might otherwise suffer in diffuscompet-ing between unconnected active sites It is now apparent that molecular architecture and mechanical properties make crucial contributions over and above the conventional chemistry at successive active sites, making it only proper to perceive such multifunctional enzymes as sophisti-cated catalytic nanomachines