Past seminars

2012/01/13

ATRIO 800

Regulatory RNA-binding proteins: from mechanistic reductionism to complete mRNA interactomes

Prof. Matthias Hentze

RNA-binding proteins (RBPs) orchestrate virtually all aspects of RNA biology. These include roles as translational regulators whose functions we have studied by means of "reductionist" biochemical approaches investigating instructive model systems. I will present our recent work on the elucidation of a new RBP-driven (Sex Lethal) regulatory mechanism that could foreshadow a more generally applying principle for mRNAs harboring upstream open reading frames. I also plan to speak about rapidly progressing new work identifying "all" mRNA binding proteins (the "mRNA interactomes") of different mammalian cells, including evidence for the existence of RNA/enzyme/metabolite (REM) networks for the coordination of cell metabolism and gene expression.

2012/01/20

ATRIO 800

Systems biology analysis of a small bacterium: lessons in humility

7th CIC bioGUNE Anniversary Lecture: Luis Serrano, PhD

Acquiring and integrating large-scale, quantitative biological data is a common feature of Systems Biology studies. However, integrating these diverse data and providing additional functional understanding on how cells work and how central processes are regulated remain an important challenge for the field of Systems Biology. A plausible approach to gaining novel biological insights from large-scale data-sets lies in the combined application of these independently developed methodologies in a suitable model organism to the same biological sample, but under different growth and stress conditions. We use Mycoplasma pneumoniae, a human pathogenic bacterium causing atypical pneumonia as model system for our study. Containing a reduced genome with only 690 ORFs, this bacterium is an ideal organism for quantitative, systems-wide studies, avoiding technical limitations due to sample complexity. We report here the metabolimics, transcriptomics and proteomics analysis of this simple organism. Our data shows that even the simplest of the bacteria has a level of complexity that prevents its full quantitative understanding.

2012/01/27

ATRIO 800

Structural studies on nascent chain ribosomal complexes. Getting ready for THE SECOND CODE

Paola Fucini, PhD

Emerging biochemical and biophysical data strongly suggest that the transformation of the potential set of proteins, encrypted in the genome of the cell, into its actual vital functional set, is a function not just of the genetic code but of an additional layer of information encoded within the newly synthesized polypeptide nascent chain (NC). To date, we know that immediately upon the first steps of protein synthesis, a variety of signals are formed within the evolving nascent chain (NC). These signals, by acting directly on the ribosome or by recruiting additional cellular machines, have the ability to dictate and direct new steps in the transformation of the NC, guiding it towards the acquisition of its final functional state and cellular location. Due to its distinct nature, I prefer to refer to this ensemble of signals as THE SECOND CODE. Despite our knowledge of its existence and importance, today we know very little about its mechanism of action. The aim of my talk is to give an overview on the latest unpublished results from our group that entail: 1) the competition/cooperation between SecA and TF on the ribosome (using the Methyl-Trosy approach, by solution state NMR) 2) the mode of binding of SecA on stalled nascent chain ribosomal complexes (using CryoEM, in collaboration with Prof. Kuhlbrandt - Max Plank Institute for Biophysics and Dr. Sean Connell - Frankfurt University, Frankfurt am Main) 3) the visualization by X-ray crystallography of a cascade of events, started by a NC mimic in the ribosomal tunnel and ending at the peptidyl transferase center, altering its configuration, to impare peptide bond formation. The results obtained have thus settled a solid research line and experimental approach to unravel the molecular determinants of the second code and to decipher how the signals, embedded in the NC, orchestrate the concerted action of the ribosome and its ancillary factors (i,e, SecA, SecB, TF and the translocon) to drive its own transformation into a functional protein.

2012/02/03

ATRIO 800

The Host Defense of Insects: A Paradigm for Innate Immunity

Prof. Jean Marc Reichhart

Professor Jean-Marc Reichhart´s group has been working since 1985 on the innate immune system. The adaptative immune system with its antibodies, B and T cells arose only once during evolution, around 500 million years ago probably in the first vertebrates. This specific immune system acts in concert with the innate immune system in roughly 45000 vertebrate species as a defence against invading microorganisms. In all invertebrates, the defence mechanisms are purely innate. In Drosophila, an infection provokes the rapid synthesis of powerful antibiotic peptides by the fat body. As an example, the basal level of expression of the antifungal peptide DROSOMYCIN, is increased a thousand fold within 30 minutes of septic injury in larvae or adults. The control of this expression involves at least two pathways that, for sake of simplicity, Professor Reichhart refers to as the TOLL and the IMD pathways. The paramount role of the TOLL and IMD pathways in the host defence of Drosophila is illustrated by experiments in which mutant flies are challenged with fungi or bacteria. In TOLL-deficient mutants, survival to fungal, but not to bacterial infection, is severely compromised. By contrast, IMD mutants are markedly affected by bacterial infections but resist fungi with a survival pattern similar to that of wild-type flies. In Vertebrates, recognition of microbes by the innate immune system takes place at the cellular level by a family of transmembrane receptors homolog to Drosophila Toll, namely the Toll like receptors (TLRs). In Drosophila however, these recognition events take place in the open circulatory system via soluble excreted recognition proteins like the peptidoglycan recognition proteins (PGRPs) and the glucan binding proteins (GNBPs). In turn, these recognition steps must be conveyed onto Toll by extracellular proteolytic signalling pathways. Upstream of the IMD pathway, other PGRPs are recognizing Gram-negative microbial determinants. Professor Jean-Marc Reichhart´s group is now interested in how these two pathways are activated and regulated.

2012/02/10

ATRIO 800

Systemic Hh coordinates the response to starvation

Jonathan Rodenfels, PhD

The ability of an organism to respond to nutritional stress is critical for its survival. Multiple conserved mechanisms have evolved to sense differences in nutrition and coordinate the organism’s response. Here, we demonstrate that midgut derived and Lipoprotein-associated Hedgehog (Hh) is systemically circulating in the hemolymph of Drosophila. Midgut Hh expression is regulated by nutrition and RNAi mediated knock-down of circulating Hedgehog leads to starvation sensitivity. Loss and gain of function experiments indicate that circulating Hh inhibits growth and developmental progression. In insects, the developmental transitions are regulated by the steroid hormone ecdysone, which is produced by prothoracic gland (PG) of the ring gland (RG). Furthermore, nutritional regulation of growth is in part mediated by the Drosophila fat body (FB).Strikingly, canonical Hedgehog pathway components are present in both tissues, the fat body and PG. In order to understand the Hedgehog mediated function during nutritional stress we ectopically activated or inhibited the Hedgehog signaling pathway specifically in the fat body or in the ring gland. Activation of the pathway in the FB phenocopies the growth inhibition observed with gain of function in circulating Hh. Moreover, pathway activation in the FB leads to a reduction in whole larval fat, glycogen and circulating sugar levels. Pathway activation in the PG results in non-pupariating larvae with strongly reduced transcription of the ecdysone biosynthetic enzyme phantom and a known ecdysone target gene, E74B. In conclusion, we propose a model whereby systemically circulating Hh regulates the organism’s response to nutritional stress by inhibiting growth and developmental progression.

2012/02/17

ATRIO 800

Understanding and controlling protein-protein interactions

Grzegorz Popowicz, PhD

Protein - protein interactions (PPI) were considered impossible to target with a drug molecule. However, recent development of both computational and experimental techniques prove that targetting PPIs is possible. We have developen a PPI oriented drug design program with a focus on Mdm2 and Mdmx oncoproteins. Our research required development of novel NMR and biophysical experiments and computational methods. This approach allowed for discovery of several scaffolds active bot in in vitro and in vivo experiments. We propose, using similar strategy for targetting novel, recently discovered PPIs.

2012/02/21

ATRIO 800

Tumor suppressor genes in cancer and beyond

Special lecture Manuel Serrano, PhD

My seminar will have three parts, each of them with unpublished data. First, I will review the current knowledge about cellular senescence and its role in tumor suppression. I will present our most recent data on the interplay between inflammation and senescence, and on the role of senescence during embryonic development. In the second part, I will present our most recent data on a new preclinical approach against lung cancer targeting the Notch pathway. This will include state-of-the-art in vivo imaging in mice. Finally, I will present our new data on the role of cell cycle regulators during the process of reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs).

2012/02/24

ATRIO 800

Structural and mechanistic studies of large macromolecules by NMR

Yiannis Gelis, PhD

Solution state NMR methodologies are routinely used to study proteins and protein complexes with molecular weights below 50kDa. Still, a combination of novel isotope labeling schemes and Methyl-TROSY has allowed obtaining high resolution structural, functional and dynamic insights to biologically important macromolecular assemblies in range of MDa. SecA is a dimeric 204 kDa protein that plays a central role in bacterial protein secretion, the fundamental cellular process by which proteins are transported across or integrated into a membrane with the expenditure of metabolic energy extracted from ATP hydrolysis. In the context of binary or ternary complexes it interacts with all the translocation components, including both the signal sequence and the mature domain of preproteins, the chaperone SecB, the protein conducting channel SecYEG and translating ribosomes. Building on methyl labeling strategies and paramagnetic spin labeling it was possible to determine the high-resolution structure of a SecA-signal sequence complex, by NMR. The recognition of signal sequences by their cognate receptors is the first and most decisive step during protein translocation. The structure, together with an extensive thermodynamic analysis and in vivo experiments revealed the details of a promiscuous recognition and the first steps of protein translocation. The same approach can be used for the study of other large macromolecular complexes such as of SecA with translating ribosomes and the Hsp90 machinery.

2012/03/02

ATRIO 800

Adipose tissue expandability, lipotoxicity and the Metabolic syndrome

Special lecture Antonio Vidal- Puig, PhD

The link between obesity and type 2 diabetes is clear on an epidemiological level, however the mechanism linking these two common disorders is not well defined. One hypothesis linking obesity to type 2 diabetes is the adipose tissue expandability hypothesis. The adipose tissue expandability hypothesis states that a failure in the capacity for adipose tissue expansion, rather than obesity per se is the key factor linking positive energy balance and type 2 diabetes. All individuals possess a maximum capacity for adipose expansion which is determined by both genetic and environmental factors. Once the adipose tissue expansion limit is reached, adipose tissue ceases to store energy efficiently and lipids begin to accumulate in other tissues. Ectopic lipid accumulation in non-adipocyte cells causes lipotoxic insults including insulin resistance, apoptosis and inflammation. This article discusses the links between adipokines, inflammation, adipose tissue expandability and lipotoxicity. Finally, we will discuss how considering the concept of allostasis may enable a better understanding of how diabetes develops and allow the rational design of new anti diabetic treatments.

2012/03/16

ATRIO 800

Transcription initiation and elongation factors compete for binding on the RNAP clamp during the transcription cycle

Finn Werner, PhD

Transcription of eukaryotic RNAPII and the closely related archaeal RNAP is facilitated by homologous transcription initiation and elongation factors. TFIIE and the archaeal homologue TFE enhance DNA strand separation during initiation by an unknown mechanism. We have developed a fluorescently labelled recombinant M. jannaschii RNAP system to probe the structure of the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE and RNAP. We have localised the position of the TFE winged helix (WH-) and Zinc-ribbon (ZR-) domains on the RNAP using single molecule FRET, and obtained information about their location relative to the nontemplate strand of the promoter using fluorescence quenching. Based on the positions of the TFE domains and the nontemplate strand (NTS) of the promoter we have developed specific structural hypotheses for the function of TFE during transcription initiation. The binding site of the TFE WH-domain on the RNAP clamp coiled coil overlaps with the binding site of the NGN domain of the universally conserved elongation factor Spt4/5 (NusG). We show that both factors compete for RNAP binding in vitro. Association of Spt4/5 with RNAP represses promoter-directed transcription in the absence of TFE, which alleviates the inhibition by displacing Spt4/5 from RNAP. During the elongation phase of transcription, the roles are reversed, as Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Despite the close resemblance to RNAPII, the archaeal transcription complex structure and protein-protein interaction network is surprisingly similar to the bacterial system. The universally conserved RNAP clamp coiled coil serves as binding site for bacterial and archaeo-eukaryotic initiation factors, region 2 of sigma 70 and the TFE WH-domain, respectively, and in addition interacts with elongation factors, NusG/RfaH and Spt4/5, respectively. However, whereas the elongation factors are evolutionary related, the initiation factors are not and the similar interaction mode is likely to be the outcome of convergent evolution. This highlights the importance of the RNAP clamp as a versatile target for factors involved in both transcription initiation and elongation.

2012/03/23

ATRIO 800

What’s new about MAT Genes and SAMe?

Special lecture Shelly C. Lu, PhD

Methionine adenosyltransferase (MAT) is the sole enzyme responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal biological methyl donor, precursor of polyamines and in the liver, precursor of glutathione. In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, 1 (forms either a dimer MAT III, or tetramer MAT I) and 2 (MAT II); while a third gene MAT2B, encodes for a regulatory subunit  that regulates MAT II. Normal differentiated liver expresses MAT1A while all extrahepatic tissues express MAT2A. MAT2A is induced in the liver during periods of rapid growth and dedifferentiation. In human hepatocellular carcinoma (HCC), MAT1A expression is reduced while MAT2A and MAT2B are induced facilitating cancer cell growth. Patients with cirrhosis have decreased MAT1A expression and inactivation of MAT I/III, culminating in decreased SAMe biosynthesis. The consequences of chronic hepatic SAMe depletion are best illustrated by the Mat1a knockout (KO) mouse model, which is characterized by increased susceptibility to steatosis and oxidative liver injury, spontaneous development of steatohepatitis and HCC. Many signal transduction pathways are altered that contribute to HCC formation in this model. These studies illustrate involvement of SAMe in numerous cellular processes; some may be independent of methylation. SAMe has been shown to be chemopreventive against HCC in rodents fed different carcinogens. Part of this may be to prevent carcinogen-induced hepatic SAMe depletion and hypomethylation. In addition, SAMe is selectively pro-apoptotic in liver cancer cells and has anti-angiogenic properties. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC. However, when tested in an in vivo HCC model, SAMe is effective in preventing HCC establishment but not as a treatment of already established HCC because the liver has compensatory mechanisms to prevent accumulation of SAMe to supraphysiologic levels. An alternate and more effective strategy is to enhance MAT1A expression in HCC. Although MAT genes are best known for SAMe biosynthesis, the fact that all three MAT genes are also found in the nucleus raises the possibility that they participate in other pathways. Indeed, MAT2B variants interact with HuR to influence its intracellular location and expression of HuR targets. Taken together, accumulating evidence show SAMe is more than just a methyl donor and MAT genes may participate in SAMe-independent intracellular processes.

2012/04/24

Atrio 801

Osteopontin, Ductular Reaction and Fibrosis

Aritz Lopategi, PhD

Fibrogenesis, or activation of the wound-healing response to persistent liver injury, is characterized by changes in the composition and quantity of extracellular matrix (ECM) deposits distorting the normal hepatic architecture by forming fibrotic scars. Failure to degrade accumulated ECM is a major reason why fibrosis progresses to cirrhosis. Emerging antifibrogenic therapies aim at inhibiting the activation of profibrogenic cells to prevent fibrillar Collagen-I deposition, degrading excessive ECM to recover normal liver architecture and restoring functional liver mass. While in normal liver, replacement of necrotic and apoptotic hepatocytes occurs by replication of quiescent adjacent hepatocytes within the lobule, when periportal hepatocytes are damaged and their division is impaired, a pool of OC arises acting as a secondary proliferative pathway. OC are bipotential cells residing primarily in the periportal region, when the liver parenchyma is damaged, OC become a source of regenerating hepatocytes, BEC and draining ductules in order to restore the functional liver mass. A by-product from the activation of this alternative proliferative pathway is the so-called ductular reaction (DR); a reactive lesion at the portal tract interface comprising small biliary ductules with the associated stroma, bile plugs and inflammatory cells. Proliferating BEC are a source of molecules that activate extracellular matrix (ECM) deposition and secrete pro-inflammatory and chemotactic cytokines, which attract and activate Hepatic stellate cells (HSC) and portal fibroblasts. These and other, yet to be discovered mediators, elicit the fibrogenic response and contribute to the progression of liver fibrosis. Although different cell types contribute to the increase in fibrillar Collagen-I during hepatic fibrogenesis, they all undergo a common process of differentiation and acquisition of a classical myofibroblast-like phenotype. HSCs are considered central ECM-producing cells within the injured liver, playing a significant role in Collagen-I deposition. In the healthy liver, they reside in the sinusoidal space of Disse; however, during chronic injury, they activate while acquiring motile, proinflammatory and profibrogenic properties. Activated HSCs migrate and accumulate at the sites of tissue repair, secreting large amounts of ECM, mostly Collagen-I and regulating ECM remodeling. Up-regulation of fibrillar Collagen-I is thus a key event leading to scarring, the pathophysiological hallmark of liver fibrosis. Though some current therapies have proven beneficial, dissecting key profibrogenic mechanisms, pathways and mediators of disease progression is vital. Several studies have identified osteopontin (OPN) as significantly up-regulated during liver injury and in HSCs. OPN is a soluble cytokine and a matrix bound protein that can remain intracellular or be secreted, hence allowing autocrine and paracrine signaling. OPN, as a matricellular phosphoglycoprotein, functions as an adaptor and modulator of cell-matrix interactions. Among its many roles, it regulates cell migration, ECM invasion and cell adhesion resulting from its ability to bind integrins and CD44. OPN expression increases in tumorigenesis, angiogenesis and in response to inflammation, cellular stress and injury. OPN plays an important role in regulating tissue remodeling and cell survival as well as in chemoattracting inflammatory cells. In this presentation we show how OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen-I protein contributing to scarring and liver fibrosis. And the contribution of OPN to the HSC profibrogenic behavior and the molecular mechanisms and signaling pathways involved in governing Collagen-I protein expression during the fibrogenic response to liver injury.

2012/04/27

ATRIO 800

PTEN restricts endothelial cell proliferation to control vessel density in Angiogenesis

Mariona Graupera, Phd

PTEN is a tumor suppressor gene, which is among the most frequently mutated in cancer. PTEN opposes the phosphoinositide-3-kinase (PI3K)/Akt pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate (PIP3). Loss of PTEN function leads to activation of the PI3K pathway, which stimulates cell growth and survival. Remarkably, emerging data show that PTEN also has PI3K independent functions. Previous studies have shown the relevance of PTEN in vascular development. However, progress in the understanding of how PTEN regulates angiogenesis has been hampered by the embryonic lethality that exhibit both the constitutive and endothelial-specific PTEN mutant mice. Hence, in order to circumvent this problem, we selectively inactivated PTEN in adult vasculature, by breeding PTEN-flox mice to tamoxifen-inducible Pdgfb-iCreER transgenic mouse line in which Cre is only active in endothelial cells after tamoxifen administration. Preliminary data obtained in postnatal retinas of PTEN-flox/Pdgfb-iCreER mice illustrates that deletion of PTEN leads to dramatic branching defects and aberrant vascular patterning of the nascent plexus. Here, we will discuss which endothelial functions are being affected upon PTEN deletion and how they are contributing to the phenotype observed. An important conclusion from our data is that the phenotype obtained upon PTEN deletion cannot be only explained by up-regulation of PIP3 levels, suggesting that PTEN also exerts lipid phosphatase independent functions in angiogenesis.

2012/05/04

ATRIO 800

Reversible ubiquitylation and the control of cell signaling

Prof. Michael Clague

Professor Michael Clague is part of the Oncology, Membrane Traffic and Signaling grouping in the Physiological Laboratory. Their research is funded by CRUK, Wellcome Trust, European Union Framework 7 and North West Cancer Research Fund. * Their over-arching interest lies in the reciprocal relationship between regulation of endocytic membrane traffic and signal transduction mechanisms. They have a long-standing interest in the links between phosphoinositide metabolism and membrane traffic. More recently they have begun to explore the role of ubiquitin in regulating the properties of endosomes and the stability of selected oncogenes. * They have a specific focus on de-ubiquitinating enzymes (DUBs). Their laboratory provided the first example of specificity to a particular type of ubiquitin chain linkage (K63, AMSH) and has provided detailed characterisation of two endosomal DUBs, which regulate down-regulation of receptor tyrosine kinases (RTKs). Currently they have projects to identify DUBs germane to cancer, which may represent attractive drug targets. * They have a particular interest in the RTK, Met, which is the receptor for hepatocyte growth/scatter factor. They are interested in how an HGF-specific signaling network is generated and how this might be disregulated in cancer. Increasingly, they are turning towards large-scale proteomics based approaches. * With regard to phosphoinositide metabolism, their current focus is on the Myotubularin family of PtdIns 3-phosphatases, some of which are mutated in heritable diseases. For example mutations in MTM1 lead to Myotubular Myopathy a form of muscular dystrophy, whilst mutations in MTMR2 lead to a neuropathological condition known as Charcot-Marie-Tooth syndrome. Their laboratory played a key role in establishing the substrate specificity of this family of enzymes.

2012/05/07

ATRIO 800

Untargeted metabolite profiling: A powerful tool for discovering metabolomic derangements that arise from genes, drugs and diseases

Prof. Steven Gross

In human patients, we often know the immediate biochemical consequence of a given inborn error of metabolism and the primary molecular target of a given drug. However, the integration of biochemical pathways often leads to complex and unanticipated consequences of gene mutations and drug actions, owing to the unrecognized interconnectivity of biochemical pathways. Untargeted plasma metabolite profiling holds enormous potential to discover, at a comprehensive systems level, how gene defects and drugs can broadly perturb complex biochemical networks and thereby give rise to unpredicted actions. Using examples, this presentation will describe how untargeted plasma metabolite profiling can be used to screen for and expand our current understanding of rare inborn errors of metabolism in neonates, as well as discover unappreciated on-target and off-target molecular mechanisms of drug actions.

2012/05/25

ATRIO 800

Combining Electron Microscopy and X-Ray techniques in Structural Biology

Jorge Navaza, PhD

Transmission Electron Microscopy (TEM) and X-Ray crystallography are two complementary techniques used in structural biology. TEM fascinates by its apparent simplicity to visualize isolated biological systems "in vitro" conditions. The observed systems are, in general, large assemblies of macromolecular structures, for example virus and viral particles. The main limitation of the technique is the rather low resolution of the produced images, usually in the nanometer range. On the other hand, X-Ray crystallography routinely determines the structures at atomic resolution of individual proteins or complexes involving a limited number of proteins. In most cases the phase problem is solved either experimentally (isomorphous replacement and related techniques) or by numerical methods where the information coming from previously determined molecular structures is efficiently used (molecular replacement). However, in the case of the very large complexes that are now crystallized, phasing by isomorphous replacement is often difficult and atomic models do not exist, in general. In this cases, EM and X-Ray data may be combined to start the process of crystal structure determination. Indeed, an initial phasing model may be a low resolution EM reconstruction of the complex used as a probe in the molecular replacement method. Phases are then extended by density modification, i.e. solvent flattening and non-crystallographic symmetry averaging. This has been done in the case of big icosahedral particles (virus) where the high symmetry is a guarantee of success in the phase extension process. Moreover, the model is usually obtained by cryo-TEM, a technique that already provides a good representation of the particle in the crystal. More recently we have applied the technique to multimeric proteins, a classic protein crystallography problem. The particles sizes were too small to use cryo-TEM so that the 3D reconstructions were performed using negatively stained samples. The main problems to solve are linked to the solvent contribution to the observed structure factors at low resolution, which is the range of resolution at which EM and X-Ray data overlap. But EM and X-Ray data may also be combined the other way: very often X-Ray crystallography determines the structures of the individual proteins that constitute the assemblies whose low resolution reconstructions were determined by TEM. It is then possible to interpret the EM image in terms of atomic models, which brings considerable complementary information to molecular biologists. This is achieved by docking individual molecules into the EM image, a technique related to molecular replacement, though a simpler one, as the role of observed structure factors is now played by the Fourier coefficients of the EM map, which provides both moduli and phases. The combined use of EM and X-Ray data will be illustrated with some applications to viral and sub-viral particles, and some multimeric proteins.

2012/06/15

ATRIO 800

Molecular characterization of TDP-43 function in vivo: cause or consequence of Amyotrophic lateral sclerosis?

Fabian Feiguin, PhD

Doctor Fabian Feiguin’s group is interested in understanding the mechanisms by which neuronal cells define and maintain axons different from dendrites and how these structures are progressively affected by degenerative diseases. For that they are using genetic, biochemical and histological approaches to identify the machinery involved in the formation of axons and dendrites in Drosophila sensitive neurons. They also utilize these tools to re-create human neurodegenerative diseases in flies. The expression of mutant human disease genes or targeting homologous genes in Drosophila confers a unique phenotype to the fly and with these fly models we are identifying the genes and mechanisms involved in progression or suppression of neuronal degeneration.

2012/06/29

Atrio 800

Legionella pneumophila - A master manipulator of host cell membrane trafficking GTPases

Matthias P. Machner, PhD

Legionella pneumophila, the causative agent of Legionnaires’ pneumonia, survives and replicates within human immune cells. The bacterium bypasses the degradative endo-lysosomal compartment and establishes a camouflaged replication vacuole (LCV) by hijacking proteins and membranes from the host cytosol. The molecular processes underlying these manipulatory events are poorly understood. Dr. Matthias P. Machner will present two studies describing how vesicle transport GTPases of the Rab family are directly targeted by L. pneumophila effector proteins during infection. The first scenario describes a cascade of events that manipulates Rab1, a regulator of vesicle trafficking to the Golgi compartment. L. pneumophila recruits and activates Rab1 through GDP/GTP exchange, and subsequently modifies Rab1 through the covalent addition of adenosine monophosphate (AMP), a process known as AMPylation, thereby locking it in the active conformation. Dr. Machner will present data showing that another effector protein named SidD catalyzes removal of AMP from Rab1, a process known as de-AMPylation, thereby priming Rab1 for inactivation and removal from the LCV membrane. In a second example Dr. Machner will describe a newly discovered interaction between a Legionella effector and the host cell GTPase Rab5, the key regulator of early endosomal fusion in eukaryotes. Their studies revealed that L. pneumophila uses Rab5 to direct an effector protein with phospholipase activity to endosomes where it alters the lipid composition of the endosomal membrane, rendering them incapable of fusion. Dr. Machner will present a model explaining how manipulation of these two Rab GTPase-regulated host pathways may allow L. pneumophila to survive and replicate within the otherwise hostile environment of a macrophage.

2012/08/02

ATRIO 801

Establishing Systems to Investigate Rack1 Signaling Complexes on the 80S Ribosome

Sean Connell, PhD

Pathological conditions like disease, cancer and bacterial/viral infection are able to modulate cellular networks that regulate protein synthesis, which, in eukaryotes, is carried out by the 80S ribosome, a large complex of RNA and proteins. This regulation of translation (activity, specificity and/or sub-cellular localization) results from the recruitment of signaling proteins to the ribosome. The protein RACK1 plays a key role in mediating these interactions by acting as a scaffolding protein to facilitate the assembly of signaling molecules into dynamic RACK1-dependent signaling complexes (RSC). We will use a variant of the split-ubiquitin yeast two-hybrid screen (SUS) to search for cellular and pathogen proteins that comprise these RSC and subsequently cryo-EM will be employed to characterize the ribosome bound by these proteins generating a '3D library' describing the RACK1 interaction network. I will present (1) on-going work in establishing the variant of the SUS and (2) results from cryo-EM investigations of ribosomal complexes that provide the framework for studying the RSC.

2012/09/14

ATRIO 800

Structure determination of genomes and genomic domains by satisfaction of spatial restraints

Marc A. Marti-Renom, PhD

The genome three-dimensional (3D) organization plays important, yet poorly understood roles in gene regulation. Chromosomes assume multiple distinct conformations in relation to the expression status of resident genes and undergo dramatic alterations in higher order structure through the cell cycle. Despite advances in microscopy, a general technique to determine the 3D conformation of chromatin has been lacking. We developed a new method for the determination of the 3D conformation of chromatin domains in the interphase nucleus, which combines 5C experiments with the computational Integrative Modeling Platform (IMP). The general approach of our method, which has been applied to study the 3D conformation of the a-globin domain in the human genome [1] and the Caulobacter crescentus whole genome [2], opens the field for comprehensive studies of the 3D conformation of chromosomal domains and contributes to a more complete characterization of genome regulation. [1] D. Baù et al. Nat Struct Mol Biol (2011) 18:107. [1] M.A. Umbarger, et al. Molecular Cell (2011) 44:252

2012/09/21

ATRIO 800

HER2 overexpression, cellular senescence and metastasis in breast cancer

Joaquín Arribas, PhD

Senescence, a terminal cell proliferation arrest, can be triggered by oncogenes. Oncogene-induced senescence is classically considered a tumor defense barrier. However, several findings show that, under certain circumstances, senescent cells may favor tumor progression because of their secretory phenotype. We have recently found that the expression in different breast epithelial cell lines of p95HER2, a constitutively active fragment of the tyrosine kinase receptor HER2, results in either increased proliferation or senescence. In senescent cells, p95HER2 elicits a secretome enriched in proteases, cytokines and growth factors. This secretory phenotype is not a mere consequence of the senescence status and requires continuous HER2 signaling to be maintained. Underscoring the functional relevance of the p95HER2-induced senescence secretome, we showed that p95HER2-induced senescent cells promotes metastasis in vivo in a non-cell autonomous manner.

2012/09/25

ATRIO 800

Rational engineering of Picornavirus capsids to produce stable vaccines

Abhay Kotecha, PhD

The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses important for human and animal health. Current picornavirus vaccines consist of intact virus capsids produced usually from inactivated virus. The inactivation may have a detrimental effect on the stability of capsids. This is an especial problem for the Foot and Mouth disease virus (FMDV) where capsids are very sensitive to pH and temperature. The viruses fall apart into pentameric assemblies below pH 6.5, presumably to facilitate uncoating after entering the host cell. Similarly, they often dissociate into pentamers at little above 37°C. This has a major consequence since one of the most important factors in the potency of inactivated virus vaccines is the integrity of virus capsids. As a result vaccines requires a cold chain and frequent immunisation of animals is required.In this project we combined in silico molecular dynamics simulation with experimental crystallographic validation and novel methods for measuring thermostabilityto rationally engineer thermo-stable FMDV capsids. We have demonstrated that in silico predictions translate into marked stabilisation of both infectious and empty viral capsids. Crystallography was used for quality assessment and to verify that the intended mutations are present and that no unanticipated structural changes have occurred as a consequence of the modifications made that might impact on the immunogenicity of the particles. We used a relatively high-throughput In-situ diffraction method to collect high-resolution datafrom micro-crystals directly from the crystallisation drops within the crystallisation plates (1). The structures of the wild type and the stabilised mutants were solved and found to be identical.The In-situ technology is a quick and easy method for collecting room temperature data, avoiding the increased mosaicityoften associated withcryo-crystallography. It also provides a contained environment faciliating safe data collection and avoiding the hazards and deleterious effects of crystal mounting. A sufficiently complete data set can often be collected form a single 96-well, diffraction-optimised plate. Stabilised capsids have been used in animal trials and found to generate increased neutralising antibody response. Such capsids will likely form the basis of the next generation of safe vaccines against the disease. (1) Axford D, et al,ActaCrystallogr D BiolCrystallogr. 2012,68:592-600.

2012/09/28

ATRIO 800

Generation of new multiserotype vaccines against bluetongue virus

Francisco Javier Ortego, PhD

Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. We have recently established in our laboratory a small animal model of BTV infection based on adult mice deficient in type I IFN receptor (IFNAR(-/-)). These mice are highly susceptible to infection with BTV. IFNAR(-/-) adult mice immunized with a BTV inactivated vaccine showed induction of neutralizing antibodies against BTV-4 and complete protection against challenge with a lethal dose of this virus suggesting that IFNAR(-/-) adult mice are a good animal model to test vaccines. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing BTV proteins. IFNAR(-/-) adult mice are being used in order to facilitate the studies of protection against the different BTV serotypes and the immune response conferred by the heterologous prime boost vaccination with DNA and rMVA expressing BTV proteins.

2012/10/05

ATRIO 800

Seminar on Molecular basis of thermoreceptors

Antonio Vicente Ferrer Montiel, PhD

Doctor Ferrer Montiel’s group focuses on understanding, in terms of structure-function, the coupling between the binding of an agonist and functional activation in thermoreceptors (TRPV1-V3 TRPM8 and TRPA1), a family of ion channels involved in signal transduction of thermal stimuli. To achieve this milestone, they use a combination of complementary approaches ranging from functional directed evolution to molecular modeling and resolution of atomic structure. Directed evolution is based on the endowment with thermal sensitivity to membranes proteins that lack it through the introduction of putative thermal sensors identified in thermoreceptors. The resulting chimeric proteins are functional assayed and domains are joined by direct and/or random mutagenesis. These studies allow us to identify the molecular determinants of thermal sensitivity and understanding how they are alosterically attached to the pore domain. As a result of these studies it is expected to design molecules that interfere with this functional coupling allowing correct pathological dysfunction of these channels. For this aim, the group has begun to obtain structural information of high resolution which unveils the molecular architecture of these channels. A priority goal is thus to solve the atomic structure of both mutants generated domains acting as temperature sensors and complete thermoreceptors. As a whole, this will have a dynamic picture of the process of "gating" channels and will contribute to the validation of new pharmacological targets.

2012/10/09

ATRIO 800

Bladder cancer progression: linking -omics, functional analyses, and biomarkers

Marta Sánchez- Carbayo, PhD

Doctor Marta Sánchez- Carbayo’s group research aims to characterize molecular mechanisms taking place in bladder cancer pathogenesis, and to translate the information of such analyses into biomarker tools that would improve the clinical management of bladder cancer patients. Their experimental strategy starts by integrating the information from the molecular characterization of bladder tumors using high-throughput approaches at the DNA, RNA and protein levels. Subsequent studies are designed: a) to understand how selected identified targets contribute to tumorigenesis and cancer progression; and b) to translate such information into multiplexed biomarkers using tissue (for disease stratification and outcome prediction) and body fluids (for early diagnosis and follow-up). More specifically, the major techniques we are currently investing on are epigenetic and proteomic approaches. Both are applied for identifying and validating individual and multiplexed biomarkers in clinical specimens, while proteomic approaches are additionally exploited for understanding molecular pathways by which genes of their interest (myopodin and KiSS-1, and others) are involved in bladder cancer. Their biomarker studies aim to address the four major clinical needs in bladder cancer: diagnosis, surveillance, progression into invasive and metastatic disease, and predict therapeutic response to the Bacille of Calmette-Guerin and cisplatin combinations. Doctor Marta Sánchez- Carbayo’s group studies have a strong translational direction, to apply the obtained results to the clinical management of bladder cancer patients. Lacking specific biomarkers and targeted therapies in routine practice in bladder cancer, they plan to exploit the information generated in high-throughput approaches to study novel candidates identified in these analyses. They characterize selected candidates differentially expressed at critical steps of bladder cancer progression identified by their previous transcriptomic analyses and integrating them with epigenetic and proteomic profiling. The functional characterization of such candidates is not only contributing to uncover mechanisms by which they may play a role in bladder cancer, but also to redefine the stages where they may be most useful as tumor markers or potential targets. In their second line of research activities, they are optimizing sample handling and protocols to implement high-throughput approaches for body fluids, and delineate multiplexed and individual biomarkers that may address the major clinical needs for bladder cancer.

2012/10/19

ATRIO 800

Seminar on the field of mammalian Prion Biology and Prion Disease

Giuseppe Legname, PhD

Giuseppe Legname completed undergraduate and postgraduate studies in biochemistry and molecular biology at the ‘Universita’ degli Studi’ of Milan, Italy. He received his Doctor of Philosophy degree in Biological Sciences from the University of Warwick in 1997 with a thesis entitled "Dianthin 30, a ribosome inactivating protein from Dianthus caryophyllus". After a long spell in Industry (Italfarmaco S.p.A.) where he co-authored many scientific papers and patents in the field of immunotherapy, he moved to Academia at the National Institute for Medical Research (NIMR), Medical Research Council in London, UK. During the three years at NIMR, his research focused on the study of the signal transduction mechanisms in thymocytes and lymphocytes, in particular thymocyte development and differentiation by transgenic animal models. The tool that he was able to establish was the first inducible expression system in the immunology of eukaryotic proteins in both cell line and transgenic animals, through which he was able to dissect the molecular requirements for thymocyte development and differentiation. This work was published in Immunity. As a follow-up of this research, he was able to make additional observations on T lymphocytes survival. These novel findings were published in the journal Science.

In 1999 he joined the faculty of the Department of Neurology at the University of California at San Francisco (UCSF), California, USA.
During the seven-and-a-half years at UCSF he was involved in basic research projects in the field of Prion Biology and Disease at the Institute for Neurodegenerative Diseases (IND), under the direction of 1997 Nobel Laureate Professor Stanley B. Prusiner.

Since December 2006, he has joined the faculty of the ‘Scuola Internazionale Superiore di Studi Avanzati’ (SISSA), in Trieste, where he is currently managing a new Prion Biology Laboratory.
At IND he was pursuing projects involving protein expression for prion detection in biological fluids and transgenic mice studies. His scientific interests included finding new tools for the study of prion diseases, such as recombinant antibodies, by using state-of-the-art recombinant DNA and phage display technology. In particular, in the last five years he provided crucial support to several projects in the prion field, including his participation in developing a sandwich Conformation-Dependent Immunoassay for early detection of prions in tissue sample and body fluids. One critical focus of the IND has been the pharmacological treatment of human prion diseases. The finding that a FDA approved drug, quinacrine, is active as anti prion drug, has spurred considerable attention as potential efficacious treatment for human prion diseases, such as Creutzfeldt-Jakob Disease (CJD) and new variant CJD. Moreover at IND he was responsible for several Projects and Science Cores of funded National Health Institute (NIH) grants on this subject. During his scientific experience in the USA he has co-authored several seminal papers, book chapters and patents. At SISSA, Giuseppe Legname has joined the Neurobiology Sector, and the main focus of his research program is in the field of mammalian Prion Biology (physiological function of the prion protein in mammals) and Prion Disease (mechanisms of prion replication and structural characterization of molecular determinants for prion infectivity).

2012/11/16

ATRIO 800

The characterization of a novel type of cellular senescence response which is elicited by complete loss of the tumor suppressor PTEN

Andrea Alimonti, PhD

Aberrant activation of oncogenes or down regulation of tumour suppressor genes trigger a premature senescence program that acts as an intrinsic tumour suppressive mechanism in vivo1. Oncogene-induced senescence (OIS) is reported to induce tumour clearance by promoting the activation of the immune response (referred as to ‘senescence surveillance’)2-4. However, whether this is a common feature of different types of senescence, still remains elusive5. Here we show that Pten loss induced cellular senescence (PICS)6, contrary to OIS, opposes tumourigenesis in absence of activation of the immune system and tumour clearance. Indeed, we find that in PICS, senescence surveillance is impaired by a senescence independent cytokine network orchestrated by activation of the signal transducer and activator of transcription 3 (Stat3). Strikingly, genetic inactivation of Stat3 in the mouse prostate epithelium reprograms the senescence associated secretory phenotype (SASP) of PICS restoring senescence surveillance. To this end, pharmacological inhibition of the Jak/Stat3 pathway also triggers an immune response, thereby activating tumour clearance. Taken together, our data demonstrate that senescence surveillance in pre-malignant senescence tumours can be blocked by the activation of senescence independent pathways. Therefore the genetic background of senescent cells should be carefully considered in order to design more effective pro-senescence therapies for cancer.

2012/11/23

ATRIO 800

Myelin membrane traffic and the role of exosome secretion in axon-glia interaction.

Eva Maria Krämer- Albers, PhD

Brain function depends on coordinated interactions between neurons and glial cells. Oligodendrocytes ensheath axons with the myelin membrane and furthermore maintain long-term axonal integrity by an unknown pathway. We are studying myelin membrane traffic and its control by axon-glia interaction. Endocytic sorting and recycling appears to be a common mode of myelin protein trafficking. The major myelin membrane protein PLP is recycling through late endosomal compartments and multivesicular bodies (MVB). MVB fusion with the oligodendroglial plasma membrane results in secretion of exosomes into the extracellular space. These exosomes carry myelin proteins in addition to genuine exosome proteins and distinct species of RNA. Intriguingly, oligodendrocyte exosome release is stimulated by electrically active neurons via neurotransmitter secretion and activation of glial neurotransmitter receptors. Moreover, neurons internalize oligodendrocyte-derived exosomes by endocytosis and recipient neurons functionally recover the exosome cargo. Functional studies indicate that the transfer of oligodendrocyte-derived exosomes protects neurons from stress and starvation. We propose that oligodendroglial exosomes participate in bidirectional neuron-glia communication and mediate the transfer of bioactive molecules from glia to neurons. The signal-mediated transfer of exosomes from glia to neurons may be implicated in neuroprotection and glial maintenance of axonal integrity.

2012/11/30

Atrio 800

Identification of novel tumor-associated antigens and improved antibody formats: the basis for new immunotherapy strategies?

Luis Álvarez-Vallina, PhD

Activating the immune system for therapeutic benefit in cancer has long been a goal in immunology and oncology. Future improvements in this cancer immunotherapy will depend on the identification on accessible targets (tumor-associated antigens) and the availability of appropriate targeting molecules. Phage display is often used as a source of recombinant antibodies against purified antigens or cell suspensions in modern biology. In our laboratory, this technology has been modified in order to obtain antibodies against native antigens in vivo. By using an ex vivo enrichment process against disaggregated tumors to purge the repertoire, and increasing the circulation time in the rounds of in vivo panning we have identified an antibody capable of mediating selective localization of phage to human prostate tumor xenografts. Affinity chromatography followed by mass spectrometric allowed the identification of the target antigen. This strategy should be applicable for the identification and validation of new targets in cancer and other human diseases. Monoclonal antibodies (mAbs) are one of the fastest growing classes of therapeutic agents. However, conventional unmodified mAbs have limitations, such as unfavorable pharmacokinetics and limited tissue penetration as they possess a defined specificity for a single antigen epitope. This is a particularly important aspect because many diseases are multifactorial, involving multiple ligands, receptors, and signaling cascades. Consequently, blockade of different pathological factors and pathways may result in improved therapeutic efficacy. Our laboratory has made great efforts for the development of the next wave of antibody-based reagents for therapy: multivalent and multispecific molecules blocking two or more relevant targets, with a format optimized for the desired pharmacokinetics and adapted to the pathological context.

2012/12/20

Parque Tecnológico de Bizkaia Edif. 101 Auditorium

Smart Composite Nanoparticles for Capture, Detection and Release

Luis M. Liz- Marzán, PhD

Determining precise diagnosis and proper therapeutic treatments for preventing and controlling pandemics and other bio-threads is central to human health. A variety of nanoparticle systems have been proposed which may even incorporate multiple functionalities that allow both detection and therapeutic actions. Metal nanoparticles in particular are of great interest because they display exceptional optical properties, related to localized surface plasmon resonances (LSPR), which give rise to well-defined absorption and scattering peaks in the visible and near-IR spectral range. Such resonances can be tuned through the size and shape of the nanoparticles, but are also extremely sensitive towards dielectric changes in the near proximity of the particles surface. Therefore, metal nanoparticles have been proposed as ideal candidates for biosensing applications. Additionally, surface plasmon resonances are characterized by large electric fields at the surface, which are responsible for the so-called surface enhanced Raman scattering (SERS) effect, which has rendered Raman spectroscopy a powerful analytical technique that allows ultrasensitive chemical or biochemical analysis, since the Raman scattering cross sections can be enhanced up to 10 orders of magnitude, so that very small amounts of analyte can be detected. In this communication, we present several examples of novel strategies to employ colloidal nanostructures comprising gold or silver in various morphologies and arrangements, as substrates for ultrasensitive detection of a wide variety of analytes, including relevant biomolecules such as prions or cancer markers, which in some cases require the design of novel techniques and composite materials for trapping them close to the metal nanostructures. Such composite nanostructures can also be foreseen as a smart system for controlled capture and release.