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Characterization of mouse NOA1 : subcellular localizaion, G-Quadruplex binding and proteolysis
(2013)
Mitochondria contain their own protein synthesis machinery with mitoribosomes that are similar to prokaryotic ribosomes. The thirteen proteins encoded in the mitochondrial genome are members of the respiratory chain complexes that generate a proton gradient, which is the electromotoric force for ATP synthesis.
NOA1 (Nitric Oxide Associated Protein-1) is a nuclear encoded GTPase that positively influences mitochondrial respiration and ATP production. Although a role in mitoribosome assembly was assigned to NOA1 the underlying molecular mechanism is poorly understood. This work shows that the multi-domain protein NOA1 serves multiple purposes for the function of mitochondria. NOA1 is a dual localized protein that makes a detour through the nucleus before mitochondrial import. The nuclear shuttling is mediated by a nuclear localization signal and the now identified nuclear export signal. SELEX (Systemic Evolution of Ligands by Exponential Enrichment) analysis revealed a G-quadruplex binding motif that characterizes NOA1 as ribonucleoprotein (RNP). G-quadruplex binding was coupled to the GTPase activity and increased the GTP hydrolysis rate. The sequence of localization events and the identification of NOA1 being a RNP lead to the discussion of an alternative import pathway for RNPs into mitochondria. The short-lived NOA1 contains ClpX recognition motifs and is specifically degraded by the mitochondrial matrix protease ClpXP. NOA1 is the first reported substrate of ClpXP in higher eukaryotes and augments the contribution of the ClpXP protease for mitochondrial metabolism. To assess the direct action of NOA1 on the mitoribosome co-sedimentation assays were performed. They showed that the interaction of NOA1 and the mitoribosome is dependent on the GTPase function and the nascent peptide chain. In vitro, NOA1 facilitated the membrane insertion of newly translated and isotope labeled mitochondrial translation products into inverted mitochondrial inner membrane vesicles. In conclusion, NOA1 is a G-quadruplex-RNP that acts as mitochondrial membrane insertion factor for mtDNA-encoded proteins.
This thesis provides a comprehensive model of the molecular function of NOA1 and is the basis for future research. The identification of NOA1 as ClpXP substrate is a major contribution to the field of mitochondrial research.
In the past century, scientists have realized that venoms are a source of a number of natural substances presenting a wide range of pharmacological properties and often displaying a high specificity for their targets. Thus, the field of toxinology came into being, which is defined as the study of toxic substances of biological origin. Toxins are found in a wide variety of animals, including fish, cone snails, scorpions, snakes, and even some mammals. To be classified as venom, these must contain substances, i.e. toxins, which disturb physiological processes and must be deliberately delivered to the target animal. Snakes have evolved one of the most sophisticated mechanisms for venom delivery. Envenomation by snakebite can induce and inhibit aggregation/agglutination of platelets as well as inhibit/activate hemostasis, but also disrupt other physiological functions via neurotoxins and angioneurin growth factors. Snake venoms contain a substantial amount of C-type lectin-related proteins (CLRPs) which are known to function, notably, as integrin inhibitors. CLRPs are heterodimers composed of homologous α and β subunits which can assemble either covalently or noncovalently to oligomers, resulting in αβ, (αβ)2 and (αβ)4 structures. Some of the main targets of CLRPs are membrane receptors, coagulation factors, and proteins essential to hemostasis. The platelet collagen receptors GPVI and α2β1 integrin as well as the von Willebrand factor receptor GPIb play important roles in platelet activation and aggregation and are considered main targets of antithrombotic drugs. In this thesis, the integrin α2β1 is particularly considered as it is the sole collagen-binding integrin on platelets. Reduced expression of this platelet receptor results in dysfunction of platelet responses. Equivalently, overexpression of α2β1 integrin results in an increased risk of thrombosis. As a result, selective inhibitors of the collagen-α2β1 interaction could give rise to effective antithrombotic drugs. Integrins are large receptors which mediate cell-cell contacts and the binding of cells to the extracellular matrix (ECM). Therefore, they play a role in physiological processes, e.g. hemostasis and immunity, as well as in pathological processes, e.g. tumor angiogenesis and atherosclerosis. 18 α and 8 β integrin subunits, with nine α subunits containing an additional A domain, associate non-covalently to form 24 heterodimers with distinct binding specificities. Integrin collagen receptors are a subclass of four receptors which all utilize the β1 subunit. The α2β1 integrin is a collagen-binding receptor expressed not only on platelets, but also on endothelial and epithelial cells. Consequently, this integrin is also essential for cell adhesion and migration playing a role in angiogenesis as well as tumor metastasis. To date, there are five known antagonists of α2β1 integrin: EMS16, rhodocetin, vixapatin, and most recently rhinocetin and flavocetin-A. The first four have been shown to be specific for the integrin α2A domain, the major collagen-binding domain. All these antagonists are CLRPs and present new leads for drug design. In the past few years, many insights into the structure and function of rhodocetin were obtained. Monoclonal antibodies proved to be advantageous in disclosing this information, making them not only useful as therapeutic agents, but also as tools for protein characterization. The venom of the Vipera palaestinae snake was recently shown to contain an α2β1 integrin inhibitor, which prevented the integrin from binding collagen. This inhibitor, called vixapatin, was the initial focus of this dissertation. Vixapatin’s interaction with the α2β1 integrin needed further characterization on a molecular and cellular level to assess its medical potential and monoclonal antibodies were to be used as a tool. Originally, vixapatin had been isolated by reversed-phase high-performance liquid chromatography. To avoid the stringency of this method, for this study, it was replaced with gentler chromatographic methods. First, the α2β1 integrin inhibitor was isolated from the crude snake venom with affinity chromatography using the α2A domain as bait, establishing a method to quickly screen venoms for α2β1-binding proteins which affect the collagenintegrin interaction. The applicability of this method to other snake venoms was shown by isolating an α2A domain-specific toxin from the venom of Trimeresurus flavoviridis. To allow further characterization of both these toxins, gel filtration and ion exchange chromatography were employed to purify the protein without the α2A domain. These classical protein purification methods resulted in similar separation patterns of both the V. palaestinae and T. flavoviridis venom proteins. Purified proteins exhibiting the potential of inhibiting integrinbinding to collagen were analyzed by two-dimensional gel electrophoresis. Both VP-i and flavocetin-A, the integrin inhibitors from V. palaestinae and T. flavoviridis, respectively, were shown to have more complex structures than was evident from the purification. Each consisted of four low-molecular-weight proteins which assembled into two bands (for VP-i) or one single band (for flavocetin-A) under non-reducing conditions. Mass spectrometry analyses revealed VP-i to belong to the family of CLRPs, just like vixapatin does. However, these two proteins differed in their primary sequences and only showed homology to one another. The toxin purified from T. flavoviridis revealed this toxin to be flavocetin-A, a heterodimeric CLRP which had so far only been shown to have GPIb-binding activity. At the time of flavocetin-A’s purification, flavocetin-B was co-purified; flavocetin-B consists of the same two α and β subunits, plus an additional γ subunit. As no sequence information is known to date for the γ subunit, it may be one of the additional proteins purified here, along with an additional δ subunit. Therefore, the toxin isolated here may actually consist of four different subunits forming a tetramer of two different heterodimers, generating an (αβ)2(γδ)2 structure. This proposed (αβ)2(γδ)2 flavocetin-A structure has binding sites for both α2β1 integrin and GPIb, with no sterical overlap, as shown by affinity chromatography using the α2A domain and the extracellular domain of the GPIb receptor. The potential of VP-i and flavocetin-A to inhibit integrin-binding to type I collagen was shown during purification: Both toxins efficiently bind to the integrin α2A domain; also, VP-i and vixapatin bind to the A domain with the same affinity. Surface plasmon resonance showed the interaction of flavocetin-A with the α2β1 integrin to be extremely strong and association to be very fast. Furthermore, both toxins were shown to inhibit binding of the wildtype integrin to collagen: VP-i and flavocetin-A acted antagonistically on cell adhesion and cell migration. Initially, the interaction between VP-i and α2β1 integrin was to be further characterized with the help of monoclonal antibodies. However, this proved problematic, the procedure requiring various optimizations. Although, after expert consultation, some monoclonal antibodies could be obtained, the cells were extremely sensitive and gave unsatisfactory results when tested as detection tools in Western blot and immunoassays. Concluding, two novel α2β1 integrin inhibitors were discovered: VP-i and flavocetin-A, which were purified using the same procedure and which have similar functions. Both are Ctype lectin-related proteins which effectively inhibit cell adhesion and migration. This underlines that nature has instrumentalized CLRPs to specifically inhibit α2β1 integrin. Further characterization of VP-i and flavocetin-A will be able to provide leads for future drug development.
Structural determinants for substrate specificity of the promiscuous multidrug efflux pump AcrB
(2013)
Opportunistic Gram-negative pathogens such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter Baumanii and Pseudomonas aeruginosa are becoming more and more multiresistant against many commonly available antibiotics [39, 40]. An important resistance mechanism of Gram-negative bacteria is the efflux of noxious compounds by tripartite systems [39, 41-44]. The best studied and most clinically relevant tripartite system is the AcrA-AcrB-TolC system of Escherichia coli, where substrate recognition and energy transduction takes place in the inner membrane protein AcrB. AcrB has a remarkably huge substrate spectrum and can recognize structurally diverse molecules, such as hexan in contrast to erythromycin, as its substrates [45]. Therefore, overproduction of the tripartite system can render a Gram-negative pathogen resistant against multiple antibiotics at once. The mechanisms of how AcrB is able to recognize such an enormous spectrum of molecules as substrates, without compromising its specificity (e.g. by neglecting essential compounds like lipids or gluclose as its susbtates), remained puzzling. Structural insight into substrate specificity was so far limited to two co-crystal structures of AcrB, where minocycline and doxorubicin, respectively, were identified bound to an internal binding pocket of AcrB. This binding pocket is particularly deeply buried into internal parts of the T monomer of AcrB and was, therefore, denoted deep binding pocket (DBP). Analysis of several AcrB co-crystal structures with substrate molecules bound to the DBP [4, 23, 25] indicated that the substrate promiscuity involved multisite binding modes within the DBP. Multisite binding modes, where different substrate molecules can bind to slightly different positions and orientations to the same binding pocket, is a common feature of multidrug recognizing proteins such as QacR or BmrR [27-29]. Nevertheless, AcrB's substrate spectrum is much broader than substrate spectra of most other multidrug recognizing proteins. Therefore, it is likely that additional mechanisms are involved in mediating the observed high substrate promiscuity of AcrB. In our recently published high-resolution AcrB/doxorubicin co-crystal structure (pdb entry: 4DX7 [23]) we were able to identify two additional substrate binding pockets in the L monomer of AcrB: i) the access pocket (AP), with an opening towards the periplasm, and ii) a putative binding site in a groove between transmembrane helices 8 and 9 (TM8/TM9 groove), accessible from the lipid layer of the inner membrane. Both binding pockets are likely to be access sites for substrates towards AcrB. Furthermore, each of the binding pockets are possibly specialized to recognize a specific subset of the entire substrate spectrum of AcrB, i.e. highly hydrophobic substrates (e.g. n-dodecyl-ß-d-maltoside or sodium dodecylsulfate) might access AcrB towards the TM8/TM9 groove and water soluble substrates (e.g. berberine) might access AcrB towards the AP. Since substrates will accumulate in the membrane or the periplasm according to their hydrophilic or hydrophobic nature, substrates will be "pre-selected" by the medium, rather than by the protein itself, and guided to their appropriate access site. This process is proposed to be called "medium- mediated pre-selection". The AcrB/doxorubicin co-crystal structure (pdb entry: 4DX7 [23]) furthermore revealed that the AP and DBP are in next neighborhood to each other and are separated by a switch loop. This switch loop adopts distinct conformations in the L, T and O monomers. Specific switch loop conformations are strongly involved in coordinating the selective occupation of both binding pockets, the AP and the DBP. The conformation of the switch loop in the L monomer (L-switch loop) opens the AP and closes the DBP, whereas the conformation of the switch loop in the T monomer (T-switch Loop) opens the DBP and closes the AP. An analysis of all asymmetric AcrB structures indicated that the L-switch loop is able to adopt multiple distinct conformations, whereas the conformation of T-switch loop remained largely congruent in all crystal structures. Moreover, each distinct switch loop conformation, observed in co-crystal structures of AcrB with occupied AP [4, 23], was perfectly adapted to the bound substrate molecule. Therefore, the putatively flexible switch loop is likely to act as an adaptive module and mediates a high binding pocket plasticity without altering the global protein structure. This binding mode is called adaptor-mediated binding mechanism, where an flexible adaptive module (like the switch loop) is able to adapt the surface shape of an binding pocket to different substrate molecules. Furthermore, structural and biochemical analyses of an AcrB G616N variant, revealed the involvement of specific switch loop conformations in the substrate specificity of AcrB. A substitution of G616, located on the switch loop, to N616 was able to alter the conformation of the switch loop exclusively in the L monomers of AcrB, whereas the switch loop conformations in T and O monomers remained congruent to the conformations observed in crystal structures of wildtype AcrB. Moreover, cells producing the AcrB G616N and MexB, both bearing the G616N amino acid substitution, exhibited a reduced resistance against certain substrates, whereas the resistance against most other substrates remained on the level of wildtype AcrB. Correlations of the phenotypes with minimal projection areas, a novel 2-spatiodimensional parameter which approximates the size of a substrate molecule, revealed that AcrB variants with a G616N substitution have a reduced efflux activity for exclusively large substrate molecules. The rejection of large substrates is most likely connected with altered L-switch loop conformations....
Biological membranes separate the cell interior from the outside and have diverse functions from signal transduction, apoptosis to transportations of ions and small molecules in and out of the cell. Most of these functions are fulfilled by proteins incorporated in the membrane. However, lipids as the main component of membrane not only serve as structural element for bilayer formation but they are also directly involved e.g. signalling processes and bilayer properties are important to mediate protein interactions. To fully understand the role of lipids, it is necessary to develop a molecular understanding of how certain membrane components modify bulk bilayer structure and dynamics. Membranes are known to have many different motions in different conditions and time scales. Temperature, pH, water content and many other conditions change membrane dynamics in a high degree. In addition to this, time scales of motions in membranes vary from ns to ms range corresponding to fast motion and slow motion, respectively. Therefore, membranes are needed to be studied systematically by varying the conditions and using methods to investigate motions in various time scales separately. The aim of this study was therefore perform a combined solid-state NMR / molecular dynamics study on model membranes. Different substrates, such as potential drugs, polarizing agents and signaling lipids were incorporated into bilayers and their location within the membrane and their effect onto the membrane was probed. NSAIDs (non-steroidal anti-inflammatory drugs), pirinixic acid derivatives, ceramides and polarizing agents were the substrates for membranes in this study. There were several experimental methods that were applied in order to investigate effects of these substrates on membrane dynamics. Different kind of phospholipids including POPC, DMPC and DPPC were used. In addition to experimental work, with the information gathered from solid state NMR experiments molecular dynamics simulations were performed to obtain more information about the membranes at the molecular level. As a result, combination of solid-state NMR with molecular dynamics simulations provides very systematic way of investigating membrane dynamics in a large range of time scales.
Pirinixic acid derivatives were special interest of this study because of their activity on peroxisome proliferator-activated receptor (PPAR) as an agonist as well as on enzymes of microsomal prostaglandin E2 synthase-1 (PGE2s) -1 and 5-lipoxygenase (5-LO) as dual inhibitor. Two potent pirinixic acid derivatives, 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoic acid (compound 2) and 2-(4-chloro-6-(quinolin-6-ylamino)pyrimidin-2-ylthio)octanoate (compound 3), have been worked and their insertion depts were investigated by combining of solid state NMR and molecular dynamics simulations. Both experimental and theoretical results pointed out that compound 3 was inserted the phospholipid bilayer more deeply than 2. NSAIDs – lipid mixtures have been also studied here. It is known that consumption of NSAIDs as in mixture with lipids results much fewer side effects than consumption of the drugs alone. Thus, it is crucial to understand interactions of NSAIDs with lipids and investigate the possible complex formation of drugs with lipids. In this study, interactions of three widely used NSAIDs, ibuprofen, diclofenac and piroxicam, with DPPC were investigated by solid-state NMR. 1H and 31P NMR results depicted that ibuprofen and diclofenac had interactions with lipids, which is an indication of drug-lipid complex formation whereas piroxicam didn’t show any interactions with lipids suggesting that no complex formation occurred in the case of piroxicam. Ceramides are known to play key roles in many cell processes and many studies showed that the functions of ceramides are related with the ceramide effects on biological membranes. Therefore, in this study, influences of ceramides on biophysics of lipid bilayers were investigated by using various solid state NMR techniques and molecular dynamics simulations. Results from molecular dynamics simulations clearly showed that ceramide and lipids have strong interactions. More evidences about ceramide-lipid interactions were provided from 1H and 14N NMR results. In addition, it was indicated by both simulation and experimental methods that ceramide increased the rigidity of DMPC by increasing chain order parameters. BTbk is a biradical, which is used as polarizing agent for dynamic nuclear polarization (DNP) experiments and found to be more efficient than other widely used polarizing agents such as TOTAPOL. Since it is a hydrophobic compound, which prefers to stay inside lipid bilayer it is important to investigate the location and orientation of bTbk along the bilayer in order to understand its enhancement profile in DNP measurements. In this study, both NMR relaxation time measurements and molecular dynamics simulations revealed that bTbk tends to stay more close to hydrophobic chain of lipids than the interfacial part of lipids at bilayer surface.
In the first part of this work, a brief introduction on lipid membranes as well as a theoretical summary on both methods of solid-state NMR and molecular dynamics simulations is given. Then, in the second part methodology is introduced for both solid-state NMR spectrometer and theoretical calculations. Afterwards, results of different membrane systems are discussed in the following parts for both solid state NMR and MD. Finally, in the last part, a summary and the conclusion of the overall results together with some future plans are explained.
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.
Retroviral vectors are powerful tools in clinical gene therapy as they integrate permanently into the target cell genome and thus guarantee long-term expression of transgenes. Therefore, they belong to the most frequently used application platforms in clinical gene therapy involving a broad range of different target cells and tissues. However, stable genomic integration of retroviral vectors can be oncogenic, as reported in several animal models and in clinical trials. In particular, γ-retroviral vectors, which derive from naturally mutagenic γ-retroviruses, integrate semirandomly into the host genome with regard to the target sequence, but have a preference for regions of active transcription and regulatory elements of transcriptionally active genes. The integration can result in overexpression of adjacent genes or disruption of ‘target’ gene expression. Moreover, γ-retroviral integration can cause modified transcripts and proteins through alternative or aberrant splicing or through premature termination of transcription.
Initially, the event of insertional mutagenesis and subsequent induction of leukemia by the genotoxicity of a γ-retroviral vector was described in a mouse model after genetic modification of hematopoietic stem cells (HSCs). Vector-related activation and overexpression of the oncogene ecotropic viral integration site-1 (Evi1) fostered clonal outgrowth and leukemogenesis. Additional genotoxic events of γ-retroviral vectors were observed in clinical HSC gene therapy trials for X-linked severe combined immune deficiency (SCID-X1), chronic granulomatous disease (X-CGD), and Wiskott-Aldrich Syndrome (WAS). But, genotoxicity induced by γ-retroviral vectors has never been described in clinical gene therapy trials involving adoptive transfer of genetically modified mature T lymphocytes. This fact is surprising, since T cells are long-lived and have a high capacity of self-renewal.
In a previous study, the susceptibility towards oncogenic transformation of mature T cells and HSCs after genetic modification was compared. It could be demonstrated that T-cell receptor (TCR)-polyclonal mature T cells are far less prone to transformation after γ-retroviral transfer of (proto-)oncogenes in vivo than HSCs. Additional experiments revealed that TCR-oligoclonal (OT-I and P14) mature T cells are transformable in the same setting and give rise to mature T-cell lymphomas (MTCLs).
In the present thesis, the susceptibility of mature T cells towards insertional mutagenesis was investigated. Within the first part of the thesis, retroviral integration sites (RISs) from 33 murine MTCLs were retrieved and subsequently analyzed in terms of integration pattern, detection of common integration sites (CIS) and gene ontology (GO). As these bioinformatic results demonstrated that insertional mutagenesis most likely contributed to mature T-cell lymphomagenesis, the susceptibility of mature T cells was directly assessed in a mouse model. Therefore, murine TCR-oligoclonal OT-I T cells were transduced with an enhanced green fluorescent protein (EGFP) encoding γ-retroviral vector and gene-modified T cells were transplanted into RAG1-/- mice. After 16 months, including one round of serial transplantation, a case of MTCL emerged. Tumor cells were characterized by CD3, CD8, TCR and ICOS expression. Integration site analysis via ligation-mediated polymerase chain reaction (LM-PCR) revealed a proviral insertion in the Janus kinase 1 (Jak1) gene. Subsequent overexpression of Jak1 could be demonstrated on transcriptional and protein level. Furthermore, T-cell lymphoma cells were characterized by an activated Jak/STAT-pathway as signal transducer and activator of transcription 3 (STAT3) was highly phosphorylated. The overexpression of Jak1 was causally implicated in tumor growth promotion as specific pharmacological inhibition of Jak1 using Ruxolitinib significantly prolonged survival of mice transplanted with these Jak1-activated tumor cells. A concluding systematic metaanalysis of available gene expression data on human mature T-cell lymphomas/leukemias confirmed the relevance of Jak/STAT overexpression in sporadic human T-cell tumorigenesis.
This was the first reported case of an insertional mutagenesis event in mature T cells in vivo. Thus, the results obtained in this thesis underline the importance of long-term monitoring of genetically modified T cells in vivo and the evaluation of vector toxicology and safety in T-cell based gene therapies. In particular, the transduction of T cells with a recombinant TCR or CAR (chimeric antigen receptor) bears a risk enhancement, as normal T-cell homeostasis is perturbed besides the general risk of insertional mutagenesis.
Eine Infektion mit dem Hepatitis B Virus (HBV) kann bei 5-10 % der infizierten Erwachsenen und 70-90 % der infizierten Kinder chronisch verlaufen. Trotz einer verfügbaren Impfung gegen die Erkrankung sind heute nach Angaben der WHO weltweit etwa 350 Mio. Menschen chronisch HBV-infiziert [Lupberger and Hildt, 2007, Hollinger and Liang, 2001]. In 5-10 % der Fälle führt eine chronische Infektion zu einer Leberfibrose und Zirrhose, welche letztlich zur Ausbildung eines hepatozellulären Karzinoms (HCC) führen kann. HCCs sind die dritthäufigste karzinomassoziierte Todesursache weltweit [Blum, 2005]. Um Therapien gegen eine HBV-Infektion und das damit erhöhte Risiko einer HCC-Entstehung entwickeln zu können, müssen die einzelnen Schritte des HBV-Replikationszyklus verstanden sein. Wesentliche Schritte der frühen Infektionsphase, insbesondere der Rezeptor bzw. Rezeptorkomplex, welcher den Zelleintritt des Virus vermittelt sowie der Transport des Virusgenoms in den Zellkern, sind bisher noch unklar. Auch der Exportprozess und die Freisetzung der Viruspartikel ist bisher noch nicht im Detail verstanden. Es ist jedoch bekannt, dass die Viruspartikel unter Nutzung der zellulären ESCRT (endosomal sorting complex required for transport)-Maschinerie aus der Zelle freigesetzt werden [Lambert et al., 2007]. Auf der Suche nach Faktoren, die in diese Vorgänge involviert sind, konnte in dieser Arbeit das vesikeltransportassoziierte Protein α-Taxilin identifiziert werden. Der Einfluss von HBV auf die α-Taxilin-Bildung und seine mögliche Beteiligung am viralen Export wurden dabei näher charakterisiert. In HBV-positiven Zellen konnte in vivo und in vitro eine signifikante Steigerung der α-Taxilin-Expression nachgewiesen werden. Diese wird hierbei durch die HBV-Proteine HBx und LHBs über den Raf/Mek/Erk-Signalweg induziert [Glatzel, 2011]. Mithilfe von knockdown-Experimenten konnte beobachtet werden, dass α-Taxilin für den Export der Viruspartikel, nicht aber für den Export subviraler Partikel (SVPs) essentiell ist. Der Export der Virionen findet hierbei über das ESCRT-System statt. Den HBV-Strukturproteinen fehlen jedoch die für die Interaktion mit dem ESCRT-System essentiellen late-Domänen. Die Proteinstruktur von α-Taxilin dagegen weist diese late-Domänen auf. In dieser Arbeit konnte diese interaktionsvermittelnde Funktion von α-Taxilin zwischen dem Virus und dem ESCRT-System charakterisiert werden. Über eine Interaktion von α-Taxilin mit dem viralen LHBs-Protein auf der einen Seite und der tsg101-Komponente des ESCRT-I-Komplexes auf der anderen Seite agiert α-Taxilin als eine Art Linker zwischen dem ESCRT-System und HBV.
Darüber hinaus wurde Annexin A5 als zellulärer Interaktionspartner für α-Taxilin identifiziert [Röttger, 2011]. Es dirigiert α-Taxilin in einer Art shuttle-Funktion auf die Zellmembran suszeptibler Zellen und bindet es an deren Zelloberfläche. Diese Exposition von α-Taxilin nimmt während der Dedifferenzierung in Korrelation mit dem Suszeptibilitätsverlust primärer Hepatozyten ab. Eine Maskierung von α-Taxilin durch eine vorherige Inkubation der Zellen mit α-Taxilin-spezifischen Antikörpern konnte die Bindung und die Aufnahme der Viren inhibieren. Überexpressionsstudien bestätigten die essentielle Rezeptorfunktion von α-Taxilin. Die verstärkte Produktion von α-Taxilin führte zur Suszeptibilität der Zellen. Auch die Speziesspezifität der Bindung zwischen humanem α-Taxilin und HBV konnte in einem Co-Immunpräzipitationsexperiment mit den rezeptorbindenden Domänen von HBV, WHV und DHBV identifiziert werden.
In der vorliegenden Arbeit konnte somit zum ersten Mal eine Rezeptorfunktion von α-Taxilin bei der Aufnahme von HBV in die Wirtszelle nachgewiesen werden. Darüber hinaus schreiben die in dieser Arbeit gemachten Beobachtungen α-Taxilin eine essentielle Funktion für die Vermittlung des ESCRT-abhängigen Exports der Virionen aus der Zelle zu. Die hierbei gewonnen Erkenntnisse sind von hoher Relevanz für die weitere Erforschung der HBV-assoziierten Pathogenese und die Etablierung eines in vivo Infektions-Modells.
Silicon wafers such as Silicon on Insulator (SOI) and strained silicon on Insulator (sSOI) are the essential and basic materials of advanced microelectronic devices. However, they often show various kinds of crystal defects which impair the function of these devices. The most efficient method to date, for detecting such defects and for determining their density, is to delineate them by etching the wafers with a suitable etching solution and characterise them via light optical microscopy. Etch pits are formed at defect sites which are etched at a faster rate than at the perfect lattice. The standard etching solution used for SOI and sSOI is a dilute version of Secco. As Secco contains carcinogenic and environmentally hazardous chromium (VI), the use of which is or will be restricted by law in many countries, suitable chromium (VI)-free etching solutions like Organic Peracid Etches (OPE), modified Chemical Polishing Etches (CP) like CP4 mod and mixtures with organic oxidizing agents like chloranil (CA) have been developed for the successful delineation of various types of crystal defects.
However there are still nanometer-sized defects which are hard to detect or escape detection by this method. Copper decoration is a well known method to magnify these defects. It consists in applying a copper nitrate solution to the back of the SOI or sSOI wafer. On annealing, copper diffuses through the substrate and the BOX (buried oxide) to the SOI/sSOI film and on quenching to room temperature, copper precipitates as copper silicide, SiCu3, foremost at crystal defects where the lattice strain is greater than at perfect lattice sites. These silicides increase the volume in these parts of the crystal lattice and defect magnification occurs. A considerable disadvantage of this method is its tendency for artefact formation, when the copper concentration used is too high, with the copper precipitating at the film surface. The consequence is a higher density of etch pits whereby true defect etch pits cannot be differentiated from those caused by artefacts.
The aim of this thesis is to show that the processes of decorating and etching can be combined successfully to delineate all crystal defects in SOI and sSOI. An ideal result would have been to find a copper decoration procedure that decorates all existing crystal defects at a copper concentration that avoids artefact formation.
In dieser Arbeit wurde das Protein OR1 ausführlich charakterisiert und die Grundlage für weitere Studien an diesem Protein gelegt. Die Zielsetzung dieser Arbeit bestand primär in der biophysikalischen Analyse eines eukaryotischen Proteorhodopsins, da bislang keine Daten zu diesen vorlagen. Dieser Ansatz ist vergleichbar mit der Studie am BR ähnlichen Rhodopsin aus dem Pilz Leptosphaeria maculans (Waschuk et al. 2005). Auch wenn man aus den Eigenschaften von OR1 keine Signatur für eukaryotische PRs herausfiltern kann, so weist OR1 eine Reihe von Charakteristika auf, die es wert sind weiterbearbeitet zu werden. Zu den hervorzuhebenden Ergebnissen dieser Arbeit zählen:
(1) OR1 zeigte in der methylotrophen Hefe Pichia pastoris ein hohes Expressionsniveau weit über der gewohnten Ausbeute bei Membranproteinen.
(2) OR1 offenbarte sich als Proteorhodopsin mit BR ähnlichen Eigenschaften wie dem niedrigen pKs-Wert des Protonenakzeptors und damit guten Protonenpumpeigenschaften über einen großen pH-Bereich. Auch bindet OR1 keinen zweiten Chromophor, was die nahen Verwandten GR und XR hingegen tun.
(3) OR1 demonstriert, dass die Konfiguration des komplexen Gegenions von Proteorhodopsinen stark variiert und sich anscheinend flexibel den physiologischen Erfordernissen des jeweiligen Organismus anpasst. In diesem Zusammenhang spielt auch das konservierte Histidin eine Rolle, da es den primären Protonenakzeptor beeinflusst. Bei OR1 stellte sich heraus, dass das Histidin den pKs Wert der D100 Position nicht signifikant beeinflusst.
(4) OR1 wurde mit 13C und 15N Atomen erfolgreich markiert und das entwickelte Protokoll für die Rekonstitution bewährte sich. Die Proteoliposomen des Wildtyps gaben sehr gut aufgelöste Festkörper-NMR Spektren. In Zukunft sind somit ausführliche NMR Studien an OR1 möglich.