Summary
There was a paradigm shift in the post-genomic era: it was recognized that the knowledge of the genomic data and of proteins encoded in the genome is not enough to understand their biological functions. Obviously, the proteome, posttranslational modifications of proteins and their dynamic regulation in complex networks are crucial for the understanding of the phenotype. A deeper, atomic level knowledge of the involved proteins and their interactions with each other as well as their low molecular weight partners is required to understand the relationship between genetically programmed and dynamically regulated protein networks. Therefore, the challenges will be to identify the components involved and to determine their molecular response mechanisms and interactions with high spatial and temporal resolution. For this purpose, different scales ranging from the atomic level to the context of cell biology are considered: dynamics of atoms determine the function of proteins, structure and dynamics of proteins shape the function of networks, dynamics of networks regulate the function of the cell.
GTP- and ATP-dependent membrane processes examined within the SFB 642 provide an excellent opportunity to close the gap between molecular and systems biology. Structures of the involved proteins, ligand binding, reaction kinetics, and protein-protein interactions are studied in order to identify the common molecular reaction mechanisms of GTP-and ATP-dependent membrane processes. Furthermore, it shall be clarified where and when proteins are inserted into the membrane or bind to it via a lipid anchor. Finally, the role of proteins and their modifications shall be investigated within biological systems such as cell cultures or animal models. To achieve this, well-known experts from different fields have formed a group and established within the SFB 642 state-of-the-art methods of structural biology, biophysics, chemical biology, systems biology, and cell biology.
The SFB 642 aims to answer important questions: which structural elements and dynamic changes of a protein are responsible for the activation of different pathways? Which multienzyme complexes are formed? What implications does the embedding of a protein within a biological system have and how is it regulated? How can the function be affected? How does the network respond to intervention with small molecules? Answering these questions will lead to a better understanding of the molecular basis of GTP- and ATP-dependent pathways and transport processes. Since mutations of proteins involved therein can cause diseases, many of the issues examined in the SFB 642 have high medical relevance. An important goal of many subprojects during the next funding period will be to move from the already detailed in vitro studies to the next higher hierarchical level of membrane processes, as linking these results with cell and system biological analyses will provides a detailed picture of signal transduction networks. On one hand, the networks of different nucleotide binding proteins shall be reconstituted on the membrane; on the other hand, experiments in the context of a living cell are planned as well.
Presentation of the research programme
Der SFB 642 will zu einem detaillierten Verständnis der molekularen Grundlagen GTP- und ATP-abhängiger Signalwege und Transportprozesse beitragen. GTP-abhängige Membranprozesse spielen eine essentielle Rolle bei der Transduktion von externen Signalen in die Zelle, sie sind somit auch zentral für die Steuerung vieler zellulärer Prozesse, wie z. B. der Zellteilung. ATP-abhängige Membranprozesse sind dagegen häufig die treibende Kraft für Transportvorgänge. Gesteuert werden GTP- und ATP-abhängige Membranprozesse durch die katalytische Hydrolyse der Nukleotide, wobei die molekularen und thermodynamischen Grundla-gen offenbar auf ähnlichen Prinzipien beruhen. Da Mutationen der beteiligten Proteine ernsthafte Erkran-kungen auslösen können, haben viele der im SFB 642 untersuchten Fragen auch eine hohe medizinische Relevanz. Daher ist es nicht nur in der Grundlagenforschung von Interesse diese Prozesse sehr detailliert zu verstehen. Ein detailliertes Verständnis wird dazu beitragen, kleine Moleküle für Therapien entwickeln zu können und die Diagnostik der Erkrankungen zu verbessern. Dies soll eine gezieltere Therapie im Rahmen der personalisierten Medizin ermöglichen.
Im SFB ergänzen sich die Expertisen auf dem Gebiet der GTP- und ATP-abhängigen Membranprozesse hervorragend. Dies wird belegt durch die hohe Zahl von insgesamt 57 gemeinsamen Publikationen allein in der vorangegangenen Förderperiode, an denen mindestens zwei Teilprojekte beteiligt waren. Dies bedeutet gegenüber dem Zeitraum 2004-2008 mit 41 gemeinsamen Publikationen noch einmal eine deutliche Steige-rung. Wir sind optimistisch, diese Zahl in der nächsten Förderperiode noch einmal steigern zu können, da sich ein sehr gut kooperierendes, sehr erfolgreiches Konsortium im SFB 642 etabliert hat.
Wie untersuchen wir die GTP- und ATP-abhängigen Prozesse im SFB 642 im Detail und was ist unser spe-zifischer Ansatz? Wir denken, dass es wichtig ist, die Proteinreaktionen räumlich und zeitlich auf verschie-denen Skalen aufzulösen: Die Dynamik der Atome bestimmt die Funktion der Proteine, die Struktur und Dynamik der Proteine die Funktion von Netzwerken und die Dynamik der Netzwerke die Funktion der Zelle. Bisher wurde ein großer Teil der Experimente in vitro durchgeführt. Ein wichtiges Ziel in der nächsten För-derperiode ist es, die Experimente näher an die In-vivo-Bedingungen heranzuführen und stärker anwen-dungsorientiert auszurichten. Daher werden viele Untersuchungen an kleinen GTPasen in der dritten För-derperiode im Kontext der Membran, in der Zelle und im Tiermodell durchgeführt. Im Bereich der ATPasen wollen wir die Expertise des SFB 642 nutzen, um auch diese Prozesse mit höchstmöglicher räumlicher und zeitlicher Auflösung zu verstehen. Es werden die Raumstrukturen der beteiligten Proteine, die Ligandenbin-dung, die Reaktionskinetiken und die Protein-Protein-Interaktionen untersucht. Es wird bestimmt, wo und wann Proteine direkt in die Membran eingelagert oder aber über Lipidanker an die Membran gebunden wer-den. Schließlich wird die Rolle der Proteine und ihrer Modifikationen im biologischen System, sowohl in Zellkulturen als auch im Tiermodell, untersucht. Die bisherigen Untersuchungen haben zu einem besseren Verständnis der molekularen Grundlagen GTP- und ATP-abhängiger Signalwege und Transportprozesse geführt. Die Orchestrierung der im SFB 642 verfügbaren Expertisen ergibt die einmalige Gelegenheit, von einem detaillierten Verständnis der molekularen Reaktionsmechanismen und Interaktionen der Proteine aus-gehend, schließlich die Integration dieser Prozesse in die Gesamtaktivität der lebenden Zelle zu verstehen. Die Expertisen und methodischen Ansätze der Wissenschaftler umspannen u. a. die Strukturbiologie, die Biophysik, die chemische Biologie, die Zellbiologie und die Systembiologie. Neben den strukturauflösenden Methoden, Röntgenstrukturanalyse und NMR-Spektroskopie stehen insbesondere zeitauflösende Methoden zur Verfügung. Die Bestimmung der drei-dimensionalen Raumstruktur ist immer ein wesentlicher Meilenstein. Unser erklärtes Ziel ist es jedoch, das präzise aufeinander abgestimmte, dynamische Wechselspiel der Proteine untereinander zu verstehen. Dazu werden u. a. zeitaufgelöste Fluoreszenz- und FTIR-Spektroskopie eingesetzt und mit biomolekularen Simulationen der Prozesse kombiniert. Eine weitere Schlüsselrolle in der Analyse spielt die chemische Biologie, die Sonden zur Verfügung stellt oder Moleküle zur Modifikation der Signalwege. Ein solch breiter Ansatz, wie wir ihn im SFB 642 verfolgen, ist für die adäquate Bearbeitung einer derart komplexen biologischen Fragestellung unerlässlich, er kann daher nicht in einer Arbeitsgruppe allein verfolgt werden. Insbesondere die Breite der methodischen Ansätze und die gebündelte Expertise auf dem Gebiet der Proteinforschung bieten den Teilprojektleitern hervorragende Forschungsbedingungen.
Durch den SFB 642 werden exzellente Arbeitsgruppen vielfältig untereinander vernetzt und die einzelnen Forschungsansätze synergistisch gebündelt. Die im SFB 642 zusammengeführte Konstellation von auf dem Gebiet der GTP- und ATP-abhängigen Membranprozesse hochrenommierten Wissenschaftlern ist im natio-nalen Vergleich einzigartig und international weithin sichtbar.
Die Teilprojekte teilen sich wie folgt auf die GTP- und die ATP-abhängigen Prozesse auf, wobei die Numme-rierung der Teilprojekte historisch gewachsen ist, da wir keine Unterteilung in Bereiche haben:
1. In TP A1 bis TP A6, TP A16, TP A17, TP A20, TP A21 und TP A24 stehen kleine G-Proteine der Ras-Superfamilie im Vordergrund, TP A11 behandelt G-Protein-gekoppelte Rezeptoren (GPCRs), die die he-terotrimeren G-Proteine aktivieren, TP A23 beschäftigt sich mit der Analyse von SRP-GTPasen.
2. TP A13, TP A19, TP A22 und TP A25 behandeln ATP-abhängige Prozesse.
3. In TP Z werden die proteomischen Untersuchungen in Zusammenarbeit mit anderen Teilprojekten durchgeführt.
Outline of the subprojects
A1 - Klaus Gerwert und Carsten Kötting
Molecular GTPase mechanisms of small and heterotrimeric G proteins
The combination of time-resolved FTIR-spectroscopy and biomolecular simulations (QM/MM) reveal reaction mechanisms of small and heterotrimeric G-proteins at atomic detail. Their interactions with GEFs, GAPs and effector proteins and their intervention by small molecules, the influence of functional water molecules and the orientation of proteins are analyzed by ATR-FTIR in the natural environment of a lipid bilayer. For the investigation of membrane-association and -dissociation also natural membranes are used. The proteins Ras, Rab and Gs and their interaction partners are analyzed in detail. The biological relevance of the results measured in vitro for in vivo conditions is validated by fluorescence lifetime measurements performed in vitro and in vivo, respectively.
A2 - Herbert Waldmann and Alfred Wittinghofer
Synthese, biophysikalische und biologische Evaluierung lipidmodifizierter Ras- und Rab-Peptide und Proteine. Identifizierung von Inhibitoren der Ras-PDEd Wechselwirkung
The proposed work will focus on the identification of small molecule inhibitors of the interactions of prenyl binding proteins like PDEd and farnesylated Ras and Rheb as a novel approach to modulate their signaling. First a biochemical assay will be established and employed to screen a ca. 200.000 member compound library. Identified hits will be used as a base for the synthesis of compounds with improved potency and selectivity. The mode of action of the inhibitors will be then investigated by means of crystal structure analysis, using biochemical, biophysical and cellular studies.
A3 - Roland Winter and Katrin Weise
Biophysical Characterization of Membrane-Associated Ras Signaling Processes
In continuation of the previous work, the complexity of the membrane-associated platform as basis for the investigation of Ras mediated signaling processes will be increased. To this end, larger membrane-associated Ras effector/regulator complexes and constructs from cellular plasma membranes will be used and their spatio-temporal organization and function analyzed. Furthermore, phosphorylation of K Ras as well as Ca2+-mediated regulators of Ras are subject of the studies on the regulation of Ras-membrane interactions. By using Rheb, a further signaling complex of the Ras family will be investigated.
A4 - Roger Goody and Yaowen Wu
Studies on the spatial and temporal distribution of Rab proteins in the cell
Modified Rab proteins will be prepared to allow their microscopic cellular location and their activation state to be determined. Application of such probes should lead to an understanding of the spatial dynamics of Rab proteins in the cell. Experiments will primarily address the mechanism of Rab targetting and its correlation with their activation. Ectopic localisation of Rab-GEFs should help to understand the role of GEFs in Rab targetting. To test models of Rab activation and localisation, the synthesis of covalent adducts between Rab proteins and GDP or GTP is planned.
A5 - Christian Herrmann
Modulation der Ras-vermittelten Signaltransduktion durch Bildung ternärer Proteinkomplexe
The novel Ras effector Nore1A displays several protein domains which allow for interaction with various proteins like tubulin and MST1 kinase and thereby for regulation of apoptosis and microtubuli formation. Malfunction of these proteins lead to the development of cancer. Molecular structures of the protein complexes will be solved and the mechanisms of interaction will be elucidated in order to understand function and regulation on the molecular level. The detailed picture obtained will enable us to design fluorescence based experiments and an optogenetic approach in order to study the way of function of the proteins in living cells.
A6 - Rolf Heumann and Raphael Stoll
Rheb enhances apoptosis - from in cell NMR spectroscopic structural and functional studies to in vivo effects of the adult mouse brain
We have recently identified a cell stress-dependent switch of Rheb signaling between the previously described growth promoting and a novel apoptosis enhancing effect. We would like to analyze whether the formation of a complex between Rheb and the redox sensitive protein kinase-1 (ASK-1) contributes to this switch mechanism. In order to pursue our structural and functional studies on Rheb in a more physiologically relevant setting we will apply in-cell NMR spectroscopic techniques to Xenopus oocytes and other cell types. We shall investigate the nucleotide dependent interaction of Rheb with Bnip3 and p75 ICD and possible posttranslational modifications thereof. A putative therapeutic relevance will be investigated in suitable mouse models of disease.
A7 (E) - Ingrid Vetter
Strukturelle Untersuchungen an der Kernpore im Kontext des durch das kleine GTP bindende Protein Ran regulierten Membrantransportes
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Under construction
A11 - Hanns Hatt
Olfactor receptor associated protein complex as regulatory element in chemosensory signaling
The project will mainly focus on the signal transduction cascade of olfactory receptor proteins in olfactory sensory neurons as well as in non-neuronal cell types. Using Live Cell Imaging, electrophysiological, pharmacological and biochemical methods we aim to identify novel olfactory receptor interaction partners and other interesting proteins playing an important physiological role in olfactory signaling. Based on our recent studies we will investigate if the activation of an olfactory receptor can trigger different signaling pathways in olfactory sensory neurons as well as in non-neuronal cell types (melanocytes, cardiomyocytes).
A13 - Ralf Erdmann
Untersuchung der ATP- und GTP-abhängigen Schritte bei der Biogenese von Peroxisomen
TP A13 addresses the dynamics, structure and function of the peroxisomal AAA-complex, which is responsible for the ATP-dependent dislocation of the import receptors during peroxisomal protein import. We intend to analyse the binding of the ATPases Pex1p/Pex6p, the core components of the AAA-complex, to the peroxisomal membrane and study their association with the peroxisomal protein import machinery. The molecular structure of the AAA-complex will be analysed by cryo-electron microscopy as well as by crystallization and X-ray analysis. Finally, the project aims to elucidate the ubiquitination cascade required for export of the import receptors and the physiological relevance of an atypically ubiquitination of specific cysteine residues of the receptor.
A15 (E) - Helmut E. Meyer
Studies of membrane-bound protein complexes and GPCR-mediated cellular processes by functional proteomics
In the focus of project A 15 are the proteomic characterization of membrane-bound protein complexes and the decoding of intracellular signaling networks. Interaction studies of peroxisomal proteins (Pex25p, Pex1p, Pex6p, Pex15p, Pex3p) aim at a better understanding of the protein import machinery and peroxisome biogenesis. Quantitative studies of the olfactory epithelium and of the sensory cilia of olfactory receptor neurons of mice shed new light onto the molecular mechanisms underlying olfaction, including models for olfactory dysfunction. Another focus lies on the characterization of the signaling cascade of the prostate-specific G-protein coupled receptor (PSGR) after odorant stimulation in a human cancer cell line using quantitative phosphoproteomics.
A16 - Philippe Bastiaens
Spatial organization of Ras signalling
Using cellular automata, we will numerically solve reaction diffusion equations in realistic cell geometries to describe molecular mechanisms that determine Ras localisation. Specifically, we will extend the dynamic models for Ras localisation to K-Ras, whose localisation is influenced by electro-static interaction with the negatively charged inner leaflet of the plasma membrane. Based on this, we will investigate how activity patterns on the cellular level arise from localised interaction networks.
A17 - Kai Sven Erdmann
Structural and functional analysis of the Lowe syndrome protein and Rab effector OCRL1
Goal of this project is to elucidate the role of the OCRL1/Rab8-module in ciliogenesis. Furthermore, a possible role of the OCRL1/Rab8 module in the generation and maintenance of epithelial cell polarization will be analyzed. Both processes could be of high relevance for the molecular understanding of Lowe syndrome. In addition, the interaction of the OCRL1 related inositol-5-phosphatase INPP5B with Rab proteins will be characterized using biophysical methods as well as principal strategies to treat Lowe syndrome will be experimentally tested.
A19 - Franz Narberhaus
In a first project, we aim at the detailed characterization of the essential function of the FtsH protease in lipopolysaccharide (LPS) biosynthesis. The underlying protein network will be identified by determining the dynamic interactome of the LPS biosynthesis enzyme LpxC and FtsH. Subsequently, biochemical studies and the E. coli Keio knockout collection will be used to characterize the function of individual components. In a second project, we will study the molecular principles of proteolysis of novel FtsH substrates by using various in vivo and in vitro approaches.
A20 - Alfred Wittinghofer
Die Rolle von Arl6 und des BBSoms beim Cilientransport
We plan to investigate the function of Arl6 in ciliary transport. Arl6 as BBS3 together with 14 other BBS genes is mutated in the ciliary disease Bardet-Biedl-Syndrom. Seven proteins encoded by BBS genes form a complex called the BBSome which is the Arl6 effector and is believed to regulate trans-port of proteins into cilia. We want to recombinantly express the BBSome in insect cells and want to investigate its interaction with Arl6 and with cargo in vitro and in vivo. Another goal is the elucidation of the structure of the full complex or various sub-complexes. Furthermore, in order to understand the regulation of this transport process we want to identify the Arl6 specific GEF and GAP.
A21 (N) - Ingrid Vetter
Mechanism of Ras-deacylation at the membrane by acyl protein thioesterases and implications for Ras signal transduction
The goal is the investigation of the regulation of membrane association of (palmitoylated) Ras by acyl protein thioesterases (APTs), especially the substrate binding modes, specificity and the activation mechanisms of the (potentially also palmitoylated) human isoenzymes APT1 and APT2. The interaction with various substrates will be functionally and structurally characterized so that rational drug design methods can help to find specific inhibitors. In addition, the mechanism of membrane extraction of Ras by APTs and the influence of the protein FKBP on this process will be explored so that the data on the in vivo-functions of the APT proteins in the cell can be correctly interpreted.
A22 (N) - Eckhard Hofmann
Transport mechanism of the bacterial ABC-transporter MsbA
In this project we will study the ATP hydrolysis in an intact ABC-transporter. To investigate this process in a native membrane context, we will apply the combination of ATR-FTIR-spectroscopy and X-ray crystallography. Of central interest is especially the coupling between the hydrolysis reaction in the NBD domain and the transport process in the transmembrane region. Structural analysis should benefit from the characterization of hydrolysis incompetent MsbA variants, which are still able to bind nucleotides.
A23 (N) - Danja Schünemann
GTP-dependent processes involved in SRP-mediated protein transport in chloroplasts
In this proposal the chloroplast SRP GTPases, cpSRP54 and the SRP receptor cpFtsY, will be analysed. The project aims at a detailed understanding of the molecular mechanisms for regulation of the cpSRP-dependent insertion of the light harvesting complex proteins into the thylakoid membrane of chloroplasts. To this end, the binding affinity and kinetics of various protein-protein interactions within the cpSRP pathway and the influence of various biological factors on the regulation of GTPase activity of the cpSRP GTPases will be investigated. In addition, we intend to analyse the molecular mechanisms of the chloroplast SRP system, that compensate the evolutionary loss of the SRP-RNA, which is conserved in all cytosolic SRP systems.
A24 (N) - Andreas Faissner
Molecular regulatory mechanisms of synaptogenesis and synaptic maturation of central nervous system neurons and of the myelination of their axons by GTPases and the up-stream nucleotide exchange factor Vav3
The objectives of the project are to elucidate the roles of the nucleotide exchange factor Vav3 and downstream small GTPases of the RhoA-family for synapse formation and activity by a combination of developmental biology and cell- and molecular biological, physiological and biochemical methods. Furthermore, to analyse the significance of these molecules for the regulation of oligodendrocyte precursor cell (OPC) migration, myelin formation and myelination of axons using a wide range of methods. The final aim is to characterize the Vav3-induced activation of small GTPases of the RhoA family with biophysical methods on the functional and on the structural level.
A25 (N) - Harald Platta
Regulation of the phosphatidylinositol 3-kinase dependent signal transduction
The class III phosphatidylinositol 3-kinase (PI3K-III) complex controls several intracellular transport pathways via its catalytic product, the signal-lipid phosphatidylinositol 3-phosphate (PtdIns3P). The cis-acting factors of the PtdIns3P-depending signal cascade will be analyzed in the context of the assembly and the regulation of the PI3K-III complex that is active in the selective autophagy of organelles. Furthermore, the PtdIns3P-binding effectors of the GTPase Cdc42 will be analyzed in the second part of the project. These trans-acting factors will be characterized regarding their possible function in different PtdIns3P-depending transport processes.
MGK - Christian Herrmann
Integrated Graduate School
In the past period of funding the SFB 642 established successfully a graduate school in order to provide a specific program of scientific education for all PhD students participating in this SFB. The program comprises lectures, scientific seminars, experimental courses and a biannual summer school organized by the fellows. By these measures networking and cooperation of the students was strongly supported as well as the generation of international contacts and cooperations. Over the past years more and more interdigitation of the SFB graduate school and the Ruhr-University Research School was reached which offers enormous support in soft skill training and in gaining interdisciplinary knowledge and experience. This cooperation will lead to a sustainable post graduate education in the field of protein research at an internationally recognised level.
Z (N) - Katja Kuhlmann, Helmut E. Meyer & Dirk Wolters
Quantitative mass spectrometry-based proteomics to analyze membrane proteins and membrane protein complexes
A detailed characterization of membrane protein complexes will be performed for peroxisomal proteins and for the AAA protease FtsH and its substrate LpxC. Our quantitative proteomics and phosphoproteomics toolbox will be used to study the influence of the GEF Vav3 on the differentiation of oligodendrocytes in cell culture. An additional aim is the identification of new phosphorylations sites in peroxisomal proteins and in OCRL1 and SDCCAG3. Furthermore, the influence of RabGGTase inhibitors on expression and prenylation of RabGTPases will be analyzed.