Cell-free expression and molecular modeling of the γ-secretase complex and G-protein-coupled receptors

Alzheimer’s disease (AD), which was first reported more than a century ago by Alhzeimer, is one of the commonest forms of dementia which affects >30 million people globally (>8 million in Europe). The origin and pathogenesis of AD is poorly understood and there is no cure available for the disease. AD is characterized by the accumulation of senile plaques composed of amyloid beta peptides (Ab 37-43) which is formed by the gamma secretase (GS) complex by cleaving amyloid precursor protein. Therefore GS can be an attractive drug target. Since GS processes several other substrates like Notch, CD44 and Cadherins, nonspecific inhibition of GS has many side effects. Due to the lack of crystal structure of GS, which is attributed to the extreme difficulties in purifying it, molecular modeling can be useful to understand its architecture. So far only low resolution cryoEM structures of the complex has been solved which only provides a rough structure of the complex at low 12-15 A resolution Furthermore the activity of GS in vitro can be achieved by means of cell-free (CF) expression. GS comprises catalytic subunits namely presenilins and supporting elements containing Pen-2, Aph-1 and Nicastrin. The origin of AD is hidden in the regulated intramembrnae proteolysis (RIP) which is involved in various physiological processes and also in leukemia. So far growth factors, cytokines, receptors, viral proteins, cell adhesion proteins, signal peptides and GS has been shown to undergo RIP. During RIP, the target proteins undergo extracellular shredding and intramembrane proteolysis. This thesis is based on molecular modeling, molecular dynamics (MD) simulations, cell-free (CF) expression, mass spectrometry, NMR, crystallization, activity assay etc of the components of GS complex and G-protein coupled receptors (GPCRs). First I validated the NMR structure of PS1 CTF in detergent micelles and lipid bilayers using coarse-grained MD simulations using MARTINI forcefield implemented in Gromacs. CTF was simulated in DPC micelles, DPPC and DLPC lipid bilayer. Starting from random configuration of detergent and lipids, micelle and lipid bilyer were formed respectively in presence of CTF and it was oriented properly to the micelle and bilyer during the simulation. Around DPC molecules formed micelle around CTF in agreement of the experimental results in which 80-85 DPC molecules are required to form micelles. The structure obtained in DPC was similar to that of NMR structure but differed in bilayer simulations showed the possibility of substrate docking in the conserved PAL motif. Simulations of CTF in implicit membrane (IMM1) in CHAMM yielded similar structure to that from coarse grained MD. I performed cell-free expression optimization, crystallization and NMR spectroscopy of Pen-2 in various detergent micelles. Additionally Pen-2 was modeled by a combination of rosetta membrane ab-initio method, HHPred distant homology modeling and incorporating NMR constraints. The models were validated by all atom and coarse grained MD simulations both in detergent micelles and POPC/DPPC lipid bilayers using MARTINI forcefield. GS operon consisting of all four subunits was co-expressed in CF and purified. The presence of of GS subunits after pull-down with Aph-1 was determined by western blotting (Pen-2) and mass spectrometry (Presenilin-1 and Aph-1). I also studied interactions of especially PS1 CTF, APP and NTF by docking and MD. I also made models and interfaces of Pen-2 with PS1 NTF and checked their stability by MD simulations and compared with experimental results. The goal is to model the interfaces between GS subunits using molecular modeling approaches based on available experimental data like cross-linking, mutations and NMR structure of C-terminal fragment of PS1 and transmembrane part of APP. The obtained interfaces of GS subunits may explain its catalysis mechanism which can be exploited for novel lead design. Due to lack of crystal/NMR structure of the GS subunits except the PS1 CTF, it is not possible to predict the effect of mutations in terms of APP cleavage. So I also developed a sequence based approach based on machine learning using support vector machine to predict the effect of PS1 CTF L383 mutations in terms of Aβ40/Aβ42 ratio with 88% accuracy. Mutational data derived from the Molgen database of Presenilin 1 mutations was using for training. GPCRs (also called 7TM receptors) form a large superfamily of membrane proteins, which can be activated by small molecules, lipids, hormones, peptides, light, pain, taste and smell etc. Although 50% of the drugs in market target GPCRs , only few are targeted therapeutically. Such wide range of targets is due to involvement of GPCRs in signaling pathways related to many diseases i.e. dementia (like Alzheimer's disease), metabolic (like diabetes) including endocrinological disorders, immunological including viral infections, cardiovascular, inflammatory, senses disorders, pain and cancer. Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) GPCRs. Docking of agonists and antagonists to CB1 and CB2 cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch, and its possible role in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB1 and CB2 receptor models were constructed based on the adenosine A2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β2AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation. Human N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in many physiological processes, including host defense against bacterial infection and resolving inflammation. The three human FPRs (FPR1, FPR2 and FPR3) share significant sequence homology and perform their action via coupling to Gi protein. Activation of FPRs induces a variety of responses, which are dependent on the agonist, cell type, receptor subtype, and also species involved. FPRs are expressed mainly by phagocytic leukocytes. Together, these receptors bind a large number of structurally diverse groups of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. For example, N-formyl-Met-Leu-Phe (fMLF), an FPR1 agonist, activates human phagocyte inflammatory responses, such as intracellular calcium mobilization, production of cytokines, generation of reactive oxygen species, and chemotaxis. This ligand can efficiently activate the major bactericidal neutrophil functions and it was one of the first characterized bacterial chemotactic peptides. Whereas fMLF is by far the most frequently used chemotactic peptide in studies of neutrophil functions, atomistic descriptions for fMLF-FPR1 binding mode are still scarce mainly because of the absence of a crystal structure of this receptor. Elucidating the binding modes may contribute to designing novel and more efficient non-peptide FPR1 drug candidates. Molecular modeling of FPR1, on the other hand, can provide an efficient way to reveal details of ligand binding and activation of the receptor. However, recent modelings of FPRs were confined only to bovine rhodopsin as a template. To locate specific ligand-receptor interactions based on a more appropriate template than rhodopsin we generated the homology models of FPR1 using the crystal structure of the chemokine receptor CXCR4, which shares over 30% sequence identity with FPR1 and is located in the same γ branch of phylogenetic tree of GPCRs (rhodopsin is located in α branch). Docking and model refinement procedures were pursued afterward. Finally, 40 ns full-atom MD simulations were conducted for the Apo form as well as for complexes of fMLF (agonist) and tBocMLF (antagonist) with FPR1 in the membrane. Based on locations of the N- and C-termini of the ligand the FPR1 extracellular pocket can be divided into two zones, namely, the anchor and activation regions. The formylated M1 residue of fMLF bound to the activation region led to a series of conformational changes of conserved residues. Internal water molecules participating in extended hydrogen bond networks were found to play a crucial role in transmitting the agonist-receptor interactions. A mechanism of initial steps of the activation concurrent with ligand binding is proposed. I accurately predicted the structure and ligand binding pose of dopamine receptor 3 (RMSD to the crystal structure: 2.13 Å) and chemokine receptor 4 (CXCR4, RMSD to the crystal structure 3.21 Å) in GPCR-Dock 2010 competition. The homology model of the dopamine receptor 3 was 8 th best overall in the competition

Time Synchronization in Multimodal Wireless Cyber-Physical Systems: A Wearable Biopotential Acquisition and Collaborative Brain-Computer Interface Paradigm

Die Forschung zu Brain-Computer Interface (BCI) hat in den letzten drei Jahren riesige Fortschritte gemacht, nicht nur im Bereich der menschlich gesteuerten Roboter, der Steuerung von Prothesen, des Interpretierens von Wörtern, der Kommunikation in einer Virtual Reality Umgebung oder der Computerspiele, sondern auch in der kognitiven Neurologie. Patienten, die unter enormen motorischen Dysfunktionen leiden (letztes Stadium Amyotrophe Lateralsklerose) könnten solch ein BCI System als alternatives Medium zur Kommunikation durch die eigene Gehirnaktivität nutzen. Neuste Studien zeigen, dass die Verwendung dieses BCI Systems in einem Gruppenexperiment helfen kann die menschliche Entscheidungstreffung deutlich zu verbessern. Dies ist ein neues Feld des BCI, nämlich das Collaborative BCI. Einerseits erfordert die Durchführung solch eines Gruppenexperiments drahtlose Hochleistungs-EEG Systeme, basierend auf BCI, welches kostengünstig und tragbar sein sollte und Langzeit-Monitoring hochwertiger EEG Daten sicherstellt. Andererseits ist es erforderlich, eine Zeitsynchronisierung zwischen den einzelnen BCI Systemen einzusetzen, damit diese für ein Gruppenexperiment zum Einsatz kommen können. Diese Herausforderungen setzten die Grundlage dieser Doktorarbeit. In dieser Arbeit wurde ein neuartiges, nicht invasives, modulares, biopotentiales Messsystem entwickelt: Dieses kann Breitband (0.5 Hz–150 Hz) Biopotentiale ableiten, bestehend aus Elektromyographie (EMG), Elektrokardiografie (EKG), Elektroencephalografie (EEG), wurde insgesamt bezeichnet als ExG bzw. das Messsystem als ExG-System benannt. Die Modularität des ExG-Systems erlaubt 8 bis hin zu 256 Kanäle zu konfigurieren, je nach Anforderung, ob in einen textilen Schlauch eingekapselt zur Erfassung von EMG Signalen, in eine textilen Weste zur Erfassung von ECG Signalen oder in eine textilen Kappe zur Erfassung von EEG Signalen. Der Einbau des ExG-Systems in eine Kappe wurde ebenfalls im Rahmen der Arbeit entwickelt. Der letzte Schritt des ExG-Systems zeigt niedriges Eingangsrauschen von 7 µVvon-Spitze-zu-Spitze und benötigt 41 mW/Kanal der Datenaufnahme im aktiven Zustand. Ein WiFi-Modul wurde für eine drahtlose Datenübertragung an einen ferngesteuerten PC in das ExG-System eingebaut. Um mit dem entwickelte System BCI Anwendungen zu ermöglichen, wurde ein akustisch und visuell evozierter Potenzialstimulator (SSVEP/AEP Stimulator) entwickelt. In eben diesem wurde ein Rasperry Pi als Zentralrechner benutzt und ein Bash basiertes Player-Skript iii einprogrammiert, das Mediadaten (Video, Audio, Ton) aus der Angabe einer Lookup Tabelle (LUT) in ihr Linux Betriebssystem spielt. Im Rahmen der Arbeit wurde eine Zeitsynchronisierung an einigen dieser ExG-Systeme mit Hilfe von einer eingebetteten Hardware/Softwarelösung durchgeführt. Die Hardwareteile bestehen aus einigen Leiterplatten, nämlich Sync Modulen mit einem Quarzoszillator, einem Mikrocontroller und einem Funkmodul (Hierbei Bluetooth 4.0). Eines von diesen ist das Sync-Addon, das mit jedem Messsystem (z.B. ExG-System) das zu synchronisieren ist, angeschlossen wird. Das andere bezeichnet man als Sync-Center, das an die Datenverarbeitungsrechner angehängt wird. Das Softwareteil übernimmt den Zeitsynchronisierungsmechanismus mit Hilfe eines funkbasierten Protokolls. Im Rahmen der Arbeit wurde ein neues energieeffizientes pairwise broadcast Zeitsynchronisationsprotokoll (PBS), welches nur theoretisch vorgestellt wurde, experimentell verifiziert. Außerdem wurde es mit anderen bestehenden Zeitsynchronisationsprotokollen auf dem aktuellen Stand der Technik evaluiert, basierend auf den Ergebnissen der gleichen Hardwareebene. In der letzten Iteration der Sync-Module wurde ein durchschnittlicher Synchronisationsfehler von 2 ms, den Konfidenzintervall von 95% berücksichtigend, erlangt. Da für Collaborative BCI, P300, ein Ereignis bezogenes Potenzial mit dem Auslöseimpuls, der 300−500 ms nach dem Vorgang eintritt, eingestellt wurde, ist die erreichte Synchronisationsgenauigkeit genügend, um solch ein Experiment durchzuführen.Brain-computer interface (BCI) has experienced the last three decades tremendous technological advances not only in the field of human controller robotics, or in controlling prosthesis, or in spelling words, or in interacting with a virtual reality environment, or in gaming but also in cognitive neuroscience. Patients suffering from severe motoric dysfunction (e.g. late stage of Amyotrophic Lateral Sclerosis) may utilise such a BCI system as an alternative medium of communication by mental activity. Recently studies have shown that usage of such BCI in a group experiment can help to improve human decision making. This is a new field of BCI, namely collaborative BCI. On one hand, performing such group experiments require wireless, high density EEG system based BCI which should be low-cost, wearable and provide long time monitoring of good quality EEG data. On the other hand time synchronization is required to be established among a group of BCI systems if they are employed for such a group experiments. These drawbacks set the foundation of this thesis work. In this work a novel non-invasive modular biopotential measurement system which can acquire wideband (0.15 Hz–200 Hz) biopotential signals consisting Electromyography (EMG), Electrocardiography (ECG), Electroencephalography (EEG) together called ExG, following ExG-system was designed. The modularity of the ExG-system allows it to be configured from 8 up to 256 channels according to the requirement if it’s to be encapsulated in a textile sleeve for recording of EMG signals, or in a textile vest for recording of ECG signals, or in a textile cap for recording of EEG signals. The assembly of the ExG-system in cap was also developed during the scope of the work. The final iteration of the ExG-system exhibits low input noise of 7 µVpeak-to-peak and require 41 mW/channel of data recording in active state. A WiFi module was embedded into the ExG-system for wireless data transmission to a remote PC. To enable the developed system for BCI applications a steady-state visually/auditory evoked potential stimulator (SSVEP/AEP stimulator) incorporating a Raspberry Pi as a main computer and a bash based player script which plays media data (video, pictures, sound) as defined in a lookup table in the Linux operating system of it. Within the scope of the work time synchronization among a group of such ExG-systems was further realized with the help of an embedded hardware/software solution. The hardware part consists of two different PCB sync modules that are incorporated with a crystal oscillator a microcontroller, a radio module (in this case Bluetooth 4.0). One of them is called the v sync-addon which is to be attached to each of the measurement systems (e.g. ExG-system) that are to be synchronized and the sync-center which is to be attached to the remote PC. On the software part, a wireless time synchronization protocol exchanging timing information among the sync-center and sync-addons must establish tight time synchronization between the ExG-system. Within the framework of this work, a novel time synchronization protocol energy efficient pairwise broadcast synchronization protocol (PBS) that was only theoretically proposed before but not evaluated on real hardware was experimentally evaluated with the developed sync modules. Moreover a benchmarking with other state-of-the-art existing time synchronization protocols based on the results from same hardware platform were drawn. In the final iteration of sync modules an average synchronization error of 2 ms was achieved considering the 95% of confidence interval. Since for collaborative BCI, P300, an event related potential was triggered with the stimuli that occur 300−500 ms after the event, the achieved synchronization accuracy is sufficient to conduct such experiments

Action of Molecular Switches in GPCRs - Theoretical and Experimental Studies

G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called “molecular switches” buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homo- and heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs

FGF-2-dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2-dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle

Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence

Coherent diffractive imaging of microtubules using an X-ray laser

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature

Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser

A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids

GPR180 is a component of TGFβ signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGF beta signalling pathway and regulates the activity of the TGF beta receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGF beta signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism.Activation of thermogenic adipocytes is a strategy to combat metabolic diseases. Here the authors report that GPR180 is a component of TGF beta signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1, and contributes to the regulation of glucose and energy metabolism