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In this thesis the integral membrane protein diacylglycerol kinase (DAGK) from E.coli is investigated with solid-state NMR. The aim is to gain an insight into the enzyme’s mechanism through integration of kinetic, structural and dynamic data. The biological function of DAGK is the transfer of the γ-phosphate group from Mg*ATP to diacylglycerol (DAG) building phosphatidic acid (PA)[6] as port of the membrane-derived oligosaccharide cycle[31,34]. Surprisingly, DAGK does not share structural or sequential similarities with other kinases[12]. Typical sequence motives found in other kinases, which catalyze phosphoryl transfer reactions, are not found[13]. In its physiological form DAGK is a homo-trimer with nine transmembrane helices, three catalytic centers and a size of 39.6 kDa.
First, the set-up of a real-time 31P MAS NMR experiment is shown. This experiment allows measuring in real-time the simultaneous ATP hydrolysis in the aqueous phase and lipid substrate phos-phorylation in the membrane phase with atomic resolution under magic angle spinning[56]. After fast transfer of the sample into the NMR spectrometer the enzymatic reaction is started with a temperature jump. This approach of real-time MAS NMR in a dual-phase system was demonstrated for the lipid substrate analogs dioleoyl- (DOG) and dibutyrylglycerol (DBG), with a C8 and C4 aliphatic chain, respectively. The combination of 31P direct and cross polarization functions as a dynamic filter. In the 31P direct polarized experiment nuclei in both phases are detected, while in the 31P cross polar-ized experiment, only nuclei in the membrane phase are detected. Rates for substrate turnover, i.e. degradation of γP-, βP, αP-ATP and build-up of βP-, αP-ADP, free phosphate as side reaction, and PA are obtained, which reveal a Michaelis-Menten behavior with regard to Mg*ATP and DBG. Here Mg*ATP and DBG follow a random-equilibrium model, where every substrate can bind indepen-dently from the other substrate. Analyses of the peak integrals from educts and products of the enzymatic reaction, revealed the stoichiometry of the reaction: 1.5 ATP molecules are used to phos-phorylate one DBG molecule. The excess of ATP is attributed to the basal ATPase activity. Further-more, experiments with ATPγS, usually regarded as a non-hydrolysable ATP-analog, where carried out. Surprisingly, DAGK hydrolyzes ATPγS and also transfers the thio-phosphate group to the lipid acceptor DBG, which points to a certain degree of plasticity in the active center. A phosphorylated enzyme intermediate was not detected. These results suggest the building of a ternary complex of Mg*ATP, DBG and DAGK performing a direct-phosphoryl transfer reaction, without passing through a phosphorylated enzyme intermediate. Experiments with the transition state analog ortho-vanadate (Vi) showed a decoupling of the ATP hydrolysis activity from lipid substrate phosphorylation. This indicates a specific transfer site for the γ-phosphate group from ATP to DAG, which can be blocked by Vi.
A general disadvantage of NMR spectroscopy compared to other spectroscopic methods is its inherent low sensitivity. One possible starting point for the improvement of signal-to-noise per unit time is the reduction of the spin-lattice relaxation time of protons[209]. Usually 95 % of the experi-mental time is required for the relaxation of the 1H to equilibrium. The addition of paramagnetic species can be used to reduce the 1H T1[233]. In a comprehensive study four different paramagnetic agents were tested: Cu2+-EDTA, Cu2+-EDTA-tag, Gd3+-TTAHA and Gd3+-DOTA. The titration of these paramagnetic complexes showed the principle feasibility of this approach, but differences between the tested species exist. The most promising complex is Gd3+-DOTA which, at a concentration of 2 mM, causes a 10-time improvement of signal-to-noise ratio per unit time. This allowed measuring 2D 13C-13C correlation spectra of proteoliposomes in one tenth of the usual required experimental time (i.e. 10 hours vs. 4 days) with good signal-to-noise.
For the investigation of structural or dynamic changes in the protein upon substrate interaction with MAS NMR, the spectral properties CP efficiency and resolution of the DAGK in liposomes needed to be improved. The most critical step during sample preparation is the reconstitution of the membrane protein from detergent micelles into a membrane of synthetic lipids under detergent removal. For this procedure the important criteria are enzymatic activity, measured in a coupled ATPase assay[55], and homogeneity of the proteoliposomes, which was tested e.g. on a discontinuous sucrose step gradient. Therefore an extensive study was carried out, in which different detergents, lipids and lipid mixtures, techniques for detergent removal and different protein-to-lipid ratios were tested. A direct correlation between high ATPase activity and good resolution was not found. Moreover, active DAGK in a mixture of DMPC and cholesterol, which emulates the membrane features of a membrane containing DAG, showed the best CP efficiency and resolution.
The assignment of the protein backbone and amino acid side chains the first mandatory step towards the investigation of structural and dynamical features influencing and defining the enzymatic mechanism by MAS NMR. As the assignment procedure is very time consuming for a total protein, a special labeling scheme for DAGK was developed, which allows assigning most of the protein areas presumably involved in enzyme catalysis. The assignment of DAGK with solution NMR[132] was not transferable to the MAS NMR spectra. Most important for the assignment process were the unique pairs[335], two consecutive amino acids which only appear once in the amino acid sequence. These unique pairs served as anchor points. Five different multinuclear MAS NMR experiments (DARR, NCO, NCA, NCACX, NCOCX) were required for the sequential assignment. It was possible to assign 35 % of the total amino acid sequence with one sample and 8 experiments acquired at 850 MHz. The secondary structure analysis showed subtle differences to the DAGK assignment with solution NMR[132], which can be attributed to the different environment in lipid bilayers and detergent micelles.
Data about structural and dynamical changes under substrate interaction can reveal details about the enzymatic mechanism. Therefore changes in chemical shift in 2D heteronuclear correlation experiments in the apo-state and under substrate saturated conditions with the substrates Mg*AMP-PNP, a non-hydrolysable ATP-analog, DOG, a mixture of Mg*AMP-PNP and DOG as well as inhibited by Vi were recorded. The most significant peak changes were observed at the interface membrane-cytoplasm as well as the the N-terminal amphipathic helix. The residues revealing chemical shift perturbations correlate with conserved residues or such residues, for which importance for catalysis and/or folding could be shown in mutation studies[8]. Especially noticeable were the changes at the amino acids Asn 72, Lys 64, His 87, Tyr 86 and Asp 95.
Beside changes of the chemical shift, changes of line width or signal doubling were observable. These changes can point to a correlation with dynamic reorientations in the μs-ms time regime, which are most relevant for enzymatic processes. The protein backbone dynamics in the apo-state as well as saturated with the substrates or inhibited with Vi were investigated with a 15N-CODEX experiment, which is based on the reorientation of the CSA tensor upon dynamical changes[350]. Specific effects of the different substrates or analogs on the protein backbone dynamic were revealed complementing the structural data and the chemical shift perturbation experiments.
The formation and maintenance of a defined three-dimensional structure is a prerequisite for most proteins in order to fulfill their function in the native context. However, there are proteins, which are intrinsically unstructured and thus natively unfolded. In addition, the misfolding and aggregation of many proteins can lead to severe diseases. The investigation of non-native states of proteins significantly contributes to the understanding of protein folding and misfolding. Nuclear magnetic resonance (NMR) spectroscopy is the only known technique that can provide information on structure and dynamics of non-native states of proteins at atomic resolution. Unfolded and non-native states of proteins have to be treated as ensembles of rapidly interconverting conformers and their observed properties are ensemble and time averaged. In this thesis, hen egg white lysozyme (HEWL) and mutants thereof have been investigated by NMR spectroscopy. The reduction of its four disulfide bridges and the successive methylation of the cysteine residues renders HEWL permanently non-native (‘HEWL-SMe’). Alternatively, the exchange of the eight cysteines for alanines results in very similar states (‘all-Ala-HEWL’). Under these conditions, HEWL-SMe and all-Ala-HEWL do not resemble random coil conformations, but exhibit residual secondary and tertiary structure. The presence of hydrophobic clusters and long-range interactions around the proteins six tryptophan residues and the modulation of these properties by single-point mutants has been observed. For the NMR spectroscopic investigation, HEWL has been isotopically labelled in E. coli by expression into inclusion bodies. After purification, the 1HN, 15NH, 13Calpha, 13Cbeta, 13C’, 1Halpha and 1Hbeta resonances of HEWL-SMe and all-Ala-HEWL have been assigned almost completely using three-dimensional NMR experiments. The analysis of secondary chemical shifts revealed regions in the proteins sequence — particularly around the six tryptophan residues—with significantly populated alpha-helix like conformations. In order to further elucidate the influence of the tryptophan side chains, a set of two new pulse sequences has been developed that allowed for the successful assignment of the 13Cg, 15Ne and 1HNe resonances in these side chains. This knowledge was eventually exploited in the interpretation of two-dimensional 15N-1H photo-CIDNP spectra, which revealed a differential solvent accessibility of the tryptophan residues in all-Ala-HEWL but not in the single point mutant W62G-all-Ala-HEWL. In addition, heteronuclear R2 relaxation rates have been determined for the indole 15Ne nuclei of all-Ala-HEWL and W62G. While in the wild-type like all-Ala-HEWL, the rates are different among the six tryptophan residues, in W62G they are more uniform. Together with relaxation data from the amide backbone, these results indicate the significant destabilization of the hydrophobic clusters in the absence of W62. In contrast, in the W108G mutant the profile of the R2 relaxation rates was not found to be significantly altered. No evidence was found by R1rho relaxation rates and relaxation dispersion measurements for conformational exchange on slower (micro- to millisecond) timescales. Residual dipolar couplings have been determined for non-native HEWL in order to retrieve structural information of these states. The differences of the W62G and the wild-type like non-native HEWL is also picked up in NH-RDCs of these proteins aligned in polyacrylamide gels. Significant positive RDCs are observed in the regions of the hydrophobic clusters in all-Ala-HEWL, but to a much lesser degree in W62G. So far, all attempts to simulate RDCs from generated non-native ensembles failed even when including long-range contacts or specific phi/psi backbone angle propensities. However, the measured RDCs can be used to cross-validate structural ensembles of non-native HEWL generated by molecular dynamics simulations that are based on restraints from the other experimental data, such as the differential solvent accessibilities from the photo-CIDNP experiments and the data on the hydrophobic clustering gained from the combined mutational and relaxation studies. Finally, non-native HEWL has been investigated for the first time using two-dimensional NMR in organic solvents, which are able to induce secondary structures and ultimately lead to amyloid formation. Under these conditions severe line broadening was observed, which was attributed to exchange between different — mostly a-helical— conformations. In summary, in this thesis methods have been developed, optimized and successfully applied for the structural and dynamical characterization of non-native states of proteins and the effect of single-point mutants on the properties of such ensembles has been investigated. Data has been gained that can considerably contribute to the further elucidation of the nature of non-native states of HEWL by molecular dynamics simulations.
Integral membrane proteins (IMPs) account for 20-40% of all open reading frames in fully sequenced genomes and they are target of approximately 60% of all modern drugs. So far, cellular expression systems are often very insufficient for the high-level production of IMPs. Toxic effects, instability or formation of inclusion bodies are frequently observed effects that prevent the synthesis of sufficient amounts of functional protein. I have successfully established an individual cell-free (CF) expression system to overcome these IMP synthesis difficulties. The CF system was established in two different expression modes. If no hydrophobic compartment is provided, the IMPs precipitate in the reaction mixture. Interestingly, these insoluble proteins are found to differ from inclusion bodies as they readily solubilize in mild detergents and the bacterial small multi drug transporter EmrE, expressed in the insoluble mode was shown to reconstitute into liposomes in an active form. Alternatively, IMPs can be synthesized in a soluble way by supplementing the CF system with detergents. A comprehensive overview of 24 commonly used detergents was provided by analyzing their impact on the CF system as well as their ability to keep three structurally very different proteins in solution. The class of long chain polyoxyethylene-alkyl-ethers turned out to be most suitable for soluble expression of a-helical EmrE, the bacterial b-barrel type nucleoside transporter Tsx and the porcine vasopressin receptor type 2, resulting in several mg of protein per mL of reaction mixture. So far IMPs have almost completely been excluded from solution nuclear magnetic resonance (NMR) analyses. I could demonstrate that CF expression enables efficient isotopic labeling of IMPs for NMR analysis and further facilitates selective labeling strategies with combinations of 13C and 15N enriched amino acids that have not been feasible before. Four different G-protein coupled receptors (GPCRs) were successfully CF expressed in preparative scale and for the human endothelin B receptor (ETB), ligand binding ability was observed. A series of truncated ETB derivatives containing nested terminal deletions have been CF produced and functionally characterized. The core area essential for Endothelin-1 binding as well as a central region responsible for ETB oligomer formation was confined to a 39 amino acid fragment including the proposed transmembrane segment 1. The binding constant (KD) of ETB was determined to 6 nM for circular ET-1 by SPR and 29 nM for linear ET-1 by TIRFS. This data indicate a large potential of the established individual CF expression system for functional IMP synthesis.
Eine große Zahl natürlicher sekundärer Metabolite sind kleine und strukturell oft sehr verschiedene Polypeptide und Polyketide. Diese bioaktiven Substanzen haben im allgemeinen ein breit aufgestelltes therapeutisches Potential und werden von verschiedenen bakteriellen Stämmen und Pilzen biosynthetisiert. Sie sind sowohl biologisch, als auch therapeutisch wichtig als Cytostatika, Immunsuppressiva und Antibiotika mit einem sehr großen antibakteriellen und antiviralen Potential. Diese oft äußerst komplexen Polypeptide und Polyketide werden von modular aufgebauten Megaenzymen in mehrstufigen Mechanismen synthetisiert. Für die Synthese dieser Peptide sind sehr große Proteincluster verantwortlich, die meistens aus einer begrenzten Anzahl sehr großer, Multidomänen umfassenden, Superenzyme aufgebaut werden. Diese Proteincluster mit einem Molekulargewicht bis in den Bereich von MegaDalton werden als nicht-ribosomale Peptidsynthetasen (NRPS) und Polyketidsynthetasen (PKS) bezeichnet. Die NRPS Systeme zeichnen sich dadurch aus, daß für die biosynthetisierten Polypeptide keine Information in Form von Nukleinsäuren wie DNA oder RNA kodiert (Walsh, C.T., 2004; Sieber & Marahiel, 2005). Für die Synthese der Polypeptide ist eine Aktivierung der einzelnen Bausteine, der Aminosäuren, durch Amino-acyl-adenylierung notwendig. Im Anschluß an die Aktivierung, wird die aktivierte Aminosäure über einen Thioester gebunden weitertransportiert. Die Thioesterbildung erfolgt an Cysteaminthiolgruppen intrinsischer 4’-Phosphopantethein-kofaktoren. Eine Modul einer NRPS stellt eine geschlossene Einheit zum Einbau einer Aminosäure mit einer hohen Spezifität für das Substrat und die biosynthetische Reaktion dar. Diese Module sind aus Domänen aufgebaut, die definierte Funktionen haben und mittels flexibler Linker miteinander verbunden sind. Die Domänen werden nach ihrer Funktion unterschieden. Die Acyl-adenylierung oder Aktivierung eines Substrates, beispielsweise einer Aminosäure, erfolgt durch die A-Domänen. Die Peptidyl- oder Acyltransportfunktion der aktivierten Substrate wird durch Thioester-domänen (T-Domäne), auch PCP (peptidyl carrier domain) genannt, bewältigt. Die Biosynthese der Kopplungsreaktion, beispielsweise die Ausbildung der Peptidbindung in NRPS Systemen, erfolgt an den Kondensations-Domänen (C-Domäne). Für die Substratspezifität eines Synthesemoduls sind die A-Domänen verantwortlich, welche die Aktivierung eines Substrat durch ATP-Hydrolyse ermöglichen. In NRPS Systemen sind auch Zyklisierungsreaktionen, durchgeführt von Cyclase-Domänen (Cy-Domänen), L/D-Epimerase-funktionen (E-Domänen) und N-Methylierungen (M-Domänen) beschrieben. So wird in Tyrocidin A an zwei Positionen spezifisch Phenylalanin in die D-Form epimerisiert und anschließend in der Peptidbiosynthese verwendet. Die Interaktion und Erkennung zwischen den multi-modularen Superenzymen, zum korrekten Aufbau der kompletten Synthetase, wurden in letzter Zeit Kommunikations-Domänen (COM-Domänen) beschrieben. Wie die aufgebaute Synthetase die korrekte Sequenz der biosynthetischen Reaktionsschritte sicherstellt ist nicht bekannt. Die enorme Diversität biosynthetischer Reaktionen in NRPS Systemen und die hohe Substratvielfalt in den verschiedensten Synthetasen unterschiedlicher Stämme eröffnet ein weites Feld für mögliche Neukombinationen von Modulen und Modifikationen von Produkten, um neue bioaktive Polypeptide mit antibiotischen Eigenschaften durch die Gestaltung neuer biosynthetischer Reaktionswege zu erhalten. Die Biosyntheseprodukte der NRPS und PKS Systeme lassen sich Gruppen kategorisieren wie Peptidantibiotika, beispielsweise beta-Lactame und makrozyklischer Polypeptide. Weitere Gruppen sind die makrozyklischen Lactone, beispielsweise Polyene und Makrolide, aromatische Verbindungen, wie Chloramphenicol, und Chinone (Tetracyclin). Die näher diskutierten Beispiele sind die antibakteriellen Polypeptide Surfactin und Tyrocidin A. Surfactin ist ein antibakteriell wirkendes makrozyklisches Lipoheptapeptid, welches von Bacillus subtilis synthetisiert wird und ein enormes antivirales Potential besitzt. Tyrocidin A ist ein antibakteriell wirkendes makrozyklisches Decapeptid und wird von Bacillus brevis und Brevisbacillus parabrevis synthetisiert. Zusätzlich werden viele bakterielle Toxine ebenfalls durch solche Systeme multi-modularer Synthetasen erzeugt. Ein Beispiel ist das Polyketid Vibriobactin, das Toxin des humanpathogenen Bakterium Vibrio cholerae. Ein zunehmendes Problem der wachsenden Weltbevölkerung moderner Gesellschaften und in den Entwicklungsländern ist die wachsende Zahl multiresistenter Bakterienstämme. Die starke Progression in der Entwicklung von Resistenzen gegen Antibiotika ist auch Gegenstand des aktuellen WHO-Reports (2006). Alarmierend ist die beschleunigte Resistenzentwicklung gegen die sogenannten Reserveantibiotika Vancomycin und Ceftazidim. Ein umfangreicheres Verständnis der Interaktion zwischen Domänen in einem Modul und zwischen Modulen eines NRPS Systems ist Grundlage für die Neukombination unterschiedlicher Module zur erfolgreichen Gestaltung neuer Biosynthesen. Da die meisten dieser Biosynthesen oder die Synthese alternativer Substanzen nicht in der Organischen Chemie zu realisieren sind oder die Produkte zu teuer wären, um diese in großen Mengen zu erzeugen, muß das Ziel sein die NRPS und PKS Systeme in ihrem modularen Aufbau und ihre Interaktion zu verstehen, um alternative Antibiotika biosynthetisch herzustellen. Peptidyl Carrier Proteine (PCPs) sind kleine zentrale Transport-Domänen, integriert in den Modulen nicht-ribosomaler Peptidsynthetasen (NRPSs). PCPs tragen kovalent über eine Phosphoesterbindung einen aus dem Protein herausragenden 4’-phosphopantetheinyl (4’-PP) Kofaktor. Der 4’-PP Kofaktor ist an der Seitenkette eines hochkonservierten Serins gebunden, welche ein zentraler Bestandteil der Phosphopantethein-Erkennungs-Sequenz ist. Die Erkennungssequenz ist homolog in vielen Proteinen mit ähnlicher Funktion, inklusive Acyl Carrier Proteinen (ACPs) der Fettsäuresynthetasen (FAS) und der Polyketidsynthetasen (PKS). Die Thiolgruppe des 4’-PP Kofaktors dient zum aktiven Transport der Substrate und der Intermediate der NRPS Systeme. Die generelle Organisation und die Kontrolle der exakt aufeinander folgenden Reaktionsschritte in der Peptidsynthetase, ist die entscheidende Frage für die Funktion des Proteinclusters (assembly line mechanism). In Modulen der NRPS Systeme folgen die PCP-Domänen C-terminal auf die Adenylierungsdomänen (A-Domäne). Die Aufgabe der A-Domänen ist die Selektion and die Aktivierung einer spezifischen Aminosäure für die „assembly line“. Die eigentliche Bildung der Peptidbindung erfolgt an der Kondensations-Domäne (C-Domäne). Der Transfer der Peptidintermediate und der aktivierten Aminosäuren zwischen A-Domänen und C-Domänen ist Aufgabe der PCPs. Um diese Funktion erfüllen zu können, ist eine große Bewegung in PCPs, bzw. des 4’-PP Kofaktors notwendig, welche als „swinging arm model“ (Weber et al., 2001) beschrieben wurde. Die PCPs koordinieren damit die Peptidbiosynthese während sie mit diversen Domänen der Synthetasen spezifisch wechselwirken müssen. Die molekularen Mechanismen des Transportes wurden bisher allerdings nicht untersucht. Eine Dynamik der Transport-Domänen wurde bereits postuliert (Kim & Prestegard, 1989; Andrec et al., 1995), konnte bisher aber nicht gezeigt werden (Weber et al., 2001). Interessanterweise zeigt sowohl apo-PCP (ohne den kovalent gebundenen 4’-PP Kofaktor) also auch holo-PCP langsamen chemischen Austausch, der als jeweils zwei stabile Konformationen beschrieben werden konnte. Diese jeweils zwei stabilen Zustände, welche sich im Austausch befinden, wurden als A und A*, für apo-PCP, und entsprechend H und H* für holo-PCP bezeichnet. Während der A- und der H-Zustand sich sowohl voneinander als auch von den entsprechenden A* und H*-Zuständen unterscheiden und spezifisch für die apo- und die holo-Form von PCP sind, ist die kalkulierte Struktur vom A*-Zustand größten Teils identisch mit der des H*-Zustandes. Die erhaltenen NMR-Strukturen des A-Zustandes, des H-Zustandes und des gemeinsamen A/H-Zustandes beschreiben in ihrer Gesamtheit ein neues Modell für ein allosterie-kontrolliertes System dualer konformationeller Zwei-Zustands-Dynamik. Zu dem beobachteten konformationellen Austausch der PCP-Domäne, konnte die Bewegung des 4’-PP Kofaktors koordiniert werden. Die Bewegung des 4’-PP Kofaktors in Verbindung mit dem konformationellen Austausch der PCP-Domäne charakterisiert die Interaktion mit katalytischen Domänen eines NRPS Moduls. Des weiteren konnte mit Hilfe des Modells die Wechselwirkung mit externen Interaktionspartnern, wie der Thioesterase II und der 4’-PP Transferase, untersucht werden. Die externe Thioesterase II der Surfactin-Synthetase (SrfTEII) von Bacillus subtilis ist ein separat expremiertes 28 KDa Protein. Sie gehört zur Familie der alpha/beta-Hydrolasen und ist verantwortlich für die Regenerierung falsch beladener 4’- PP Kofaktoren der Peptidyl Carrier Domänen. Die SrfTEII wurde mittels Lösungs-NMR untersucht, die Resonanzen wurden zugeordnet, erste strukturelle Modelle konnte berechnet werden und das Interaktionsverhalten mit verschiedenen modifizierten Kofaktoren und PCPs wurde analysiert. Die Spezifität der Substraterkennung durch die SrfTEII kann beschrieben werden. Interessanterweise zeigt auch die SrfTEII Doppelpeaks für einzelne Aminosäuren, diese können als Indikator für eine spezifische Substraterkennung durch das Enzym verwendet werden und helfen den funktionellen Unterschied zwischen der SrfTEI-Domäne und SrfTEII zu verstehen.
According to the World Health Organization (WHO) bacterial resistance to antibiotic drug therapy is emerging as a major public health problem around the world. Infectious diseases seriously threaten the health and economy of all countries. Hence, the preservation of the effectiveness of antibiotics is a world wide priority. The key to preserving the power of antibiotics lies in maintaining their diversity. Many microorganisms are capable of producing these bioactive products, the so called antibiotics. Specifically in microorganisms, polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) produce these natural bioactive compounds. Besides being used as antibiotics these non-ribosomal peptides and polyketides display an even broader spectrum of biological activities, e.g. as antivirals, immunosuppressants or in antitumor therapy. The wide functional spectrum of the peptides and ketides is due to their structural diversity. Mostly they are cyclic or branched cyclic compounds, containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications such as epimerization, methylation, N‐formylation or heterocyclization. It is has been shown that these modifications are important for biological activity, but little is known about their biosynthetic origin.
PKS and NRPS are multidomain protein assembly lines which function by sequentially elongating a growing polyketide or peptide chain by incorporating acyl units or amino acids, respectively. The growing product is attached via a thioester linkage to the 4’-phosphopantetheine (4’-Ppant) arm of a holo acyl carrier protein (ACP) in PKSs or holo peptidyl carrier protein (PCP) in NRPSs and is passed from one module to another along the chain of reaction centers. The modular arrangement makes PKS and NRPS systems an interesting target for protein engineering. More than 200 novel polyketide compounds have already been created by module swapping, gene deletion or other specific manipulations. Unfortunately, however, engineered PKS often fail to produce significant amounts of the desired products. Structural studies may faciliate yield improvement from engineered systems by providing a more complete understanding of the interface between the different domains. While some information about domain-domain interactions, involving the most common enzymatic modules, ketosynthase and acyltransferase, is starting to emerge, little is known about the interaction of ACP domains with other modifying enzymes such as methyltransferases, epimerases or halogenases.
To further improve the understanding of domain-domain interactions this work focuses on the curacin A assembly line. Curacin A, which exhibits anti-mitotic activity, is from the marine cyanobacterium Lyngbya majuscula. This outstanding natural product contains a cyclopropane ring, a thiazoline ring, an internal cis double bond and a terminal alkene. The biosynthesis of curacin A is performed by a 2.2 Mega Dalton (MDa) hybrid PKS-NRPS cluster. A 10-enzyme assembly catalyzes the formation of the cyclopropane moiety as the first building block of the final product. Interestingly, for these enzymes the substrate is presented by an unusual cluster of three consecutive ACPs (ACPI,II,III). Little is known about the function of multiple ACPs which are supposed to increase the overall flux for enhanced production of secondary metabolites.
The first task in this work was to elucidate the structural effect of the triplet ACP repetition by nuclear magnetic resonance (NMR). The initial data show that the excised ACPI, ACPII or ACPIII proteins resulted in [15N, 1H]-TROSY spectra with strong chemical shift perturbations (CSPs), suggesting an effect on the structure. The triplet ACP domains display a high sequence identity (93- 100%) making structural investigation using usual NMR techniques due to high peak overlap impossible. To enable the investigation of the triplet ACP in its native composition we developed a powerful method, the three fragment ligation. Segmental labeling allows incorporating isotopes into one single domain in its multidomain context. As a result we could prepare the triplet ACP with only one domain isotopically labeled and therefore assign the full length protein. In this way our method paved the way to study the structural effects of the triplet ACP repetition. We could show unexpectedly, that, despite the fact that the triplet repeat of CurA ACPI,II,III has a synergistic effect in the biosynthesis of CurA, the domains are structurally independent.
In the second part of this work, we studied the structure of the isolated ACPI domain. Our results show that the CurA ACPI undergoes no major conformational changes upon activation via phosphopantetheinylation and therefore contradicts the conformational switching model which has been proposed for PCPs. Further we report the NMR solution structures of holo-ACPI and 3-hydroxyl-3-methylglutaryl (HMG)-ACPI. Data obtained from filtered nuclear overhauser effect (NOE) experiments indicate that the substrate HMG is not sequestered but presented on the ACP surface.
In the third part of this work we focussed on the protein-protein interactions of the isolated ACPI with its cognate interaction partners. We were especially interested in the interaction with the halogenase (Cur Hal), the first enzyme within the curacin A sub-cluster, acting on the initial hydroxyl-methyl-glutaryl (HMG) attached to ACPI. Primarily we studied the interaction using NMR titration and fluorescence anisotropy measurements. Surprisingly no complex between ACPI and Cur Hal could be detected. The combination of an activity assay using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and mutational analysis revealed several amino acids of ACPI that strongly decrease the activity of CurA Hal. Mapping these mutations according to their effect on the Cur Hal activity onto the structure of HMG-ACPI displays that these amino acids surround the substrate and form a consecutive surface. These results suggest that this surface is important for Cur Hal recognition and selectivity. Our research presented herein is an excellent example for protein-protein interactions in PKS systems underlying a specific recognition process.
Die Fähigkeit der spezifischen und kontextabhängigen zellulären Adaption auf intrinsische und/oder extrinsische Signale ist das Fundament zellulärer Homöostase. Verschiedene Signale werden von Membranrezeptoren oder intrazellulären Rezeptoren erkannt und ermöglichen die molekulare Anpassung zellulärer Prozesse. Komplexe, ineinandergreifende Proteinnetzwerke sind dabei elementar in der Regulation der Zelle. Proteine und deren Funktionen werden dabei nach Bedarf reguliert und unterliegen einem ständigen proteolytischen Umsatz.
Die stimulusabhängige Gentranskription und/oder Proteintranslation nimmt hier eine zentrale Stellung ein, da die zugrundeliegende Maschinerie die Komposition und Funktion der Proteinnetzwerke entsprechend anpassen kann. Zusätzlich zur Regulation der Proteinabundanz werden Proteine posttranslational modifiziert, um deren Eigenschaften rasch zu ändern. Zu posttranslationalen Modifikationen zählen die Ubiquitinierung und/oder Phosphorylierung, welche die Proteinfunktionen hochdynamisch regulieren. Deregulierte Proteinnetzwerke werden oft mit Neurodegeneration und Autoimmun- oder Krebserkrankungen assoziiert. Auch Infektionen mit humanpathogenen Bakterien greifen stark in den Regulierungsprozess von Proteinnetzwerken und deren Funktionen ein. Die zelluläre Homöostase wird dadurch herausgefordert.
Bakterien der Gattung Salmonella sind zoonotische, gramnegative, fakultativ intrazelluläre Pathogene, welche weltweit millionenfach Salmonellen-erkrankungen hervorrufen. Von besonderer Bedeutung ist dabei Salmonella enterica serovar Typhimurium (hiernach Salmonella), welches im Menschen, meist durch mangelnde Hygienemaßnahmen, Gastroenteritis auslöst.
Immunität in Epithelzellen wird über das angeborene Immunsystem vermittelt und dient der Pathogenerkennung und -bekämpfung. Die Toll-like Rezeptoren (TLR) gehören zu den Mustererkennungsrezeptoren (pattern recognition receptors), welche spezifische mikrobielle Strukturen detektieren und eine kontextabhängige zelluläre Antwort generieren. Danger-Rezeptoren erkennen hingegen nicht direkt das Pathogen, sondern zelluläre Perturbationen, welche durch Zellschäden oder bakterielle Invasionen verursacht werden. Die intrinsische Fähigkeit der Wirtszelle, sich gegen Infektionen/Gefahren zu wehren wird dabei als zellautonome Immunität bezeichnet. Dabei nehmen induzierte proinflammatorische Signalwege und zelluläre Stressantworten eine wichtige Stellung ein. Die zelluläre Stressantwort aktiviert unter anderem die selektive Autophagie. Diese kann spezifisch aberrante Organelle, Proteine und invasive Pathogene abbauen. Ein weiterer Stresssignalweg ist die integrated stress response (ISR), welche eine selektive Proteintranslation erlaubt und damit die Auflösung des proteintoxischen Stresses ermöglicht.
Zur Penetration von Epithelzellen benötigt Salmonella ein komplexes System an Virulenzfaktoren, welches die bakterielle Internalisierung und Proliferation in der Wirtszelle ermöglicht. Salmonella nutzt dazu ein Typ-III-Sekretionssystem. Das System sekretiert bakterielle Virulenzfaktoren in die Zelle, sodass eine hochspezifische Modulierung des Wirtes erzwungen wird.
Die Virulenzfaktoren SopE und SopE2 spielen dabei eine Schlüsselrolle, da sie die Pathogenität von Salmonella maßgeblich vermitteln. Durch molekulare Mimikry von Wirts GTP (Guanosintriphosphat) -Austauschfaktoren aktivieren SopE und SopE2 die Rho GTPasen CDC42 und Rac1. GTP-geladenes CDC42 und Rac1 wiederum aktivieren das Aktinzytoskelett und stimulieren die Polymerisierung von Aktinfilamenten über den Arp2/3-Komplex an der Invasionsstelle. Das Pathogen wird dadurch in ein membranumhülltes Vesikel, die sogenannte Salmonella-containing Vakuole (SCV), aufgenommen. Die SCV stellt eine protektive, replikative, intrazelluläre Nische des Pathogens dar und wird permanent durch verschiedene Virulenzfaktoren moduliert.
Im Allgemeinen führt die Aktivierung von Mustererkennungsrezeptoren und Danger-Rezeptoren also zu einer zellulären Stressantwort und Entzündungsreaktion, wodurch es zur Bekämpfung der Infektion kommt. Inflammatorische Signalwege werden meist über den zentralen Transkriptionsfaktor NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) vermittelt. NF-κB bewirkt die Induktion von proinflammatorischen Effektoren und Stressgenen. Zellautonome Immunität wird zusätzlich durch antibakterielle Autophagie ermöglicht, wobei Salmonella selektiv über das lysosomale System abgebaut werden. Das bakterielle Typ-III-Sekretionssystem verursacht an einigen wenigen SCVs Membranschäden, sodass Salmonella das Wirtszytosol penetrieren. Zytosolische Bakterien werden dabei spezifisch ubiquitiniert. Dies erlaubt die Erkennung durch die Autophagie-Maschinerie.
In der vorliegenden Arbeit wurde die zellautonome Immunität von Epithelzellen während einer akuten Salmonella Infektion durch quantitative Proteomik untersucht...
Post-translational modifications (PTMs) of cell fate regulating proteins determine their stability, localization and function and control the activation of cell protective signaling pathways. Particularly in aberrantly dividing cancer cells the surveillance of cell cycle progression is essential to control tumorigenicity. In a variety of carcinomas, lymphomas and leukemias, the tumor-suppressive functions of the apoptosis- and senescence-regulating promyelocytic leukemia protein (PML) is controlled by numerous PTMs. PML poly-ubiquitylation and polySUMOylation at several lysine (K) residues induce PML degradation that is correlated to a progressive and invasive cancer phenotype. Besides several known E3 ubiquitin protein ligases that are involved in PML degradation, less is known about PML-specific deubiquitylases (DUBs), the respective DUB-controlled ubiquitin conjugation sites and the functional consequences of PML (de)ubiquitylation. Here, we show that the pro-tumorigenic DUB USP22 critically regulates PML protein stability by modifying PML residue K394 in advanced colon carcinoma cells in vitro and that this modification also impacts the homeostasis and function of the leukemia-associated mutant variant PML-RARα. We found that ablation of USP22 decreases PML mono-ubiquitylation and correlates with a prolonged protein half-live in colon carcinoma and acute promyelocytic leukemia (APL) cell lines. Additionally, silencing of USP22 enhances interferon and interferon-stimulated gene (ISG) expression in APL cells in vitro, which together with prolonged PML-RARα stability increases the APL cell sensitivity towards differentiation treatment. In accordance with the novel roles of USP22 as suppressor of the interferon response in human intestinal epithelial cells (hIECs), our findings imply USP22-dependent surveillance of PML-RARα stability and interferon signaling in human leukemia cells, revealing USP22 as central regulator of leukemia pathogenesis.
5-LO is the key enzyme in the biosynthesis of proinflammatory leukotrienes, converting arachidonic acid to 5-HPETE, and in a second step 5-HPETE to leukotriene A4. Although the 5-LO promoter possesses characteristics of so called housekeeping genes, such as lack of TATA/CCAAT boxes and existence of several Sp1 binding sites, the 5 -LO gene is tissue specifically expressed in primarily immune competent cells of myeloid origin including granulocytes, monocytes, macrophages, mast cells and B-lymphocytes. 5-LO gene expression in MM6 and HL-60 cells is strongly induced after differentiation of the cells with TGF-beta and 1,25(OH)2D3. In some monocytic cancer cell lines, such as HL-60 TB and U937, TGF-beta and 1,25(OH)2D3 treatment are not able to activate 5-LO gene transcription. It was demonstrated, that in these cell lines the 5-LO core promoter is heavily methylated and that only demethylation by the DNA methyltransferase inhibitor 5-aza-2 deoxycytidine (Adc) upregulated the 5-LO mRNA levels. It was also shown that the histone deacetylase inhibitor TsA could induce 5-LO mRNA levels, but only in 1,25(OH)2D3/TGF-beta inducible MM6 cells. Interestingly the 1,25(OH)2D3/TGF-beta effect on 5-LO expression is reduced, when combined with TsA. Reporter gene assays revealed that 5-LO promoter activity is strongly induced after 24 h treatment with 330 nM TsA (construct N10 up to 35 fold in HeLa cells). The effect is dependent on the presence of the proximal Sp1 binding site GC4 (-53 bp to –48 bp in relation to the major TIS) in both HeLa and MM6 cells. In vitro binding of the transcription factor Sp1 to this site has been demonstrated in gel shift assays and DNase I footprints. Mutation of the binding site resulted in a loss of basal promoter activity in both 5-LO negative HeLa cells and in 5-LO positive MM6 cells, as well as in the loss of TsA inducibility. The mutational study of different Sp1 binding sites in a larger promoter context revealed the interaction or respectively the additive effect of the multiple Sp1 binding sites of the 5-LO promoter on basal as well as on TsA upregulated promoter activity. However, GC4 seems to be of special relevance for both the basal promoter activity, possibly recruiting the basal transcription machinery, as well as for the TsA induced upregulation of 5-LO promoter activity. TsA does not alter the protein expression levels of Sp1 and Sp3 as investigated in Western blot analysis, neither in HeLa nor in MM6 cells. DNA affinity purification assays revealed that TsA had no effect on the DNA affinity of Sp1 or Sp3. In vitro binding of both Sp1 and Sp3 to the 5-fold GC box, GC4 and GC5 was demonstrated by DAPA analysis, but histone deacetylase inhibition did not change the associated protein amounts. Finally, in vivo binding of Sp1 and Sp3 was investigated in chromatin immunoprecipitation assay (ChIP) in MM6 cells. TsA clearly induced the association of both proteins to the promoter area surrounding the TIS. Upon TsA treatment also RNA polymerase II binding to the area surrounding the TIS (-318 to +52 bp) was increased and even initiated in the more distal promoter parts –1049 to –292 bp, which are negatively regulated in reporter gene assays. Interestingly histone H4 is already highly acetylated without TsA treatment and the acetylation status of H4 remains unchanged after histone deacetylase inhibition, indicating an open chromatin structure of the 5-LO gene in MM6 cells. In a cotransfection study with Sp1 and Sp3, the transactivating potential of factors was investigated and in accordance with the ChIP data, Sp1 and Sp3 increased the promoter activity, but only after TsA treatment. In gel shift assays, the influence of DNA methylation on Sp1 binding was investigated. The results indicate different roles for the three proximal promoter sites. Whereas Sp1 binding to the 5-fold GC box and GC4 is impaired by DNA methylation, binding to GC5 is even increased. A cotransfection study with methylated 5-LO promoter constructs and the murine methyl-CpG binding proteins suggest MBD1 involvement in the regulation of the 5-LO promoter. Since in gel shifts Sp1 binding is inhibited by DNA methylation, at least to the 5-fold GC box and the activating element GC4, and similarly the mutation/deletion of the same sites strongly reduces or inhibits promoter activity, it is likely to assume, that the loss of promoter activity after in vitro methylation is in the first place due to impaired Sp1/Sp3 binding. Together the data underline the importance and complexity of Sp1/Sp3 binding to the GC rich sites in the regulation of 5-LO promoter activity in response to the histone deacetylase inhibitor TsA as well as in respect to DNA methylation.
Die chromosomale Translokation t(4;11) ist mit einer aggressiven pro-B ALL im Kleinkindesalter assoziiert und stellt eine der häufigsten genetischen Veränderungen des MLL Gens dar. Bei bis zu 40 % der untersuchten Translokationen des MLL Gens wurde das AF4 Gen als Translokationspartner identifiziert. Durch Arbeiten in unserer Arbeitsgruppe konnte in Focus Formation Experimenten das wachstumstrans-formierende Potenzial sowohl des Wildtyp AF4 Proteins, als auch des bei der Translokation entstehenden AF4•MLL Fusionsproteins, nachgewiesen werden. Es kann somit als gesichert angesehen werden, daß es sich bei dem Wildtyp-AF4 Protein um ein Proto-Onkoprotein und bei dem AF4•MLL Fusionsprotein um ein Onkoprotein handelt. Der für beide Proteine identische Bereich beschränkt sich auf die ersten 360 Aminosäuren des AF4 Proteins, was der Hypothese führte, daß der N-Terminale Bereich des AF4 Proteins (AF4•N) für das beobachtete onkogene Potential in murinen embryonalen Fibroblasten verantwortlich ist. Ein mit dem AF4•N Protein durchgeführter Hefe-2-Hybrid Screen identifizierte die beiden E3-Ligasen SIAH1 und SIAH2 als Bindungspartner. Hierbei handelt es sich um Tumorsupressor- Proteine, die durch Ubiquitinylierung von Zielproteinen diese dem proteasomalen Abbau zuführen. Unter normalen physiologischen Bedingungen unterliegt das AF4 Protein einem raschen Abbau am Proteasom. Dies ist für das AF4•MLL Fusionsprotein nur noch eingeschränkt möglich, da es wie für das Wildtyp-MLL beobachetet proteolytisch gespalten wird, mit sich selbst dimerisiert und dann nicht mehr über das Proteasom abgebaut werden kann. Eine Bindung der beiden E3-Ligasen SIAH1 und SIAH2 konnte jedoch noch beobachtet werden, deshalb sollte die AF4 und SIAH Protein-Protein-Interaktion genauer untersucht werden. Hierzu wurden Hefe-2-Hybrid Experimente mit Deletionsmutanten durchgeführt, um die minimalen Kontakt-domänen zu identifiziert. Die Stärke der Interaktionen wurde durch ß-Galaktosidasetests ermittelt. Die identifizierte minimale AF4 Proteindomäne enthält das für die Erkennung durch die E3-Ligasen notwendige PxAxVxP Motiv und hat eine Länge von 25 Aminosäuren. Für die E3-Ligasen SIAH1 und SIAH2 konnte der für die Interaktion notwendige Kontaktbereich innerhalb der sogenannten Substrat-Bindungs-Domäne (SBD) lokalisiert werden. Interessanterweise ist nicht die große Furche des Dimerisierungsinterfaces der beiden SIAH Monomere der Kontaktbereich, sondern der proximale Zink-Finger Bereich. Die experimentell ermittelten Proteindomänen wurden in geeignete bakterielle Expressionssysteme kloniert und ihre in vitro Interaktion durch Pulldown-Experimente bestätigt. Die strukturelle Aufklärung der Kontaktdomäne erfolgte dann mit Hilfe der NMR-Fast-Mapping Methode. Mit dieser kombinatorischen Methode wurden die an der AF4 Bindung beteiligten Aminosäuren des SIAH Proteins durch Änderung ihrer chemischen Verschiebung im [15N,1H] HSQC-Spektrum nach Titration mit steigenden AF4 Konzentrationen identifiziert. Aus den erhaltenen Daten und anhand der bekannten SIAH Röntgenstruktur konnte ein Modell für die Bindung des AF4 Proteins an die E3-Ligase SIAH1 erstellt werden. Über die Funktion des Proto-Onkoproteins AF4 ist bis dato wenig bekannt. Es gibt Hinweise, daß alle Vertreter der ALF Proteinfamilie über transkriptionsaktivierende Eigenschaften verfügen. Da posttranslationale Modifikationen von Proteinen, wie z.B. Sumoylierung, häufig zur Regulation von Transkriptionsfaktoren beobachtet werden, wurden Untersuchungen auf posttranslationale Modifikationen des AF4 Proteins durchgeführt. Hierzu wurde durch Mutation der E3-Ligase Erkennungssequenz PxAxVxP eine stabilisierte AF4 Mutante hergestellt. Durch Immunopräzipitations Experimente nach Transfektion in 293T Zellen konnte sowohl die Sumoylierung, als auch Tyrosin Phosphorylierungen des AF4 Proteins nachgewiesen werden.
The evolution of cell-free protein synthesis (CFPS) over recent decades has made it a widely used system for expressing membrane proteins (MPs). Unlike traditional methods, CFPS allows direct and translocon-independent expression of MPs within lipid membranes, such as liposomes or nanodiscs (NDs), without the need for detergent solubilization. This open nature of CF systems enables customization of the experimental environment, including expression conditions, choice of nanoparticles (NPs), lipid composition, and addition of stabilizing molecules.
Membrane scaffold protein (MSP)-based NDs emerged as a gold standard for cotranslational solubilization of MPs using the CF-system. This approach allowed not only biochemical characterization, but also structural studies of MPs and even GPCRs. However, to solubilize MPs inside nanoparticles via the traditional reconstitution route, apart from MSPs other scaffolds were successfully implemented, e.g. the saposin A (commercially known as Salipro) scaffold system or the synthetic styrene maleic acid lipid particles (SMALPs). In this study the potential of saposin A-based nanoparticles (SapNPs) was explored for cotranslational MP solubilization.
Three strategies for applying SapNPs in CF systems were investigated: preassembly, (i) coassembly (ii), and coexpression (iii). (i) Preassembly involved forming SapNPs before CF expression and adding them to the CF reaction. In coassembly mode SapA and lipids were mixed in the CF reaction for spontaneous assembly with the synthesized MP. In coexpression mode lipids were added to the CF reaction while coexpressing SapA with the MP target. Proteorhodopsin (PR) served as a model protein to evaluate these strategies due to its ability to oligomerize and straightforward quantification using the cofactor retinal. Preassembled SapNPs provided homogeneous, aggregate-free particles yielding up to 200 µM solubilized PR inside in the CF reaction. Coassembly was also successfully applied to produce PR/SapNP complexes at slightly lower yields, however the system was prone to produce soluble aggregates at too high PR template concentrations and overall needed more adjustments. Coexpression resulted in PR yields below 20 µM and was not considered viable for MP production. Finally, the preassembled SapNPs were used to produce functional G-protein coupled receptor probes. Despite lower overall performance compared to MSP-based systems, SapNPs showed potential as an alternative in CF systems for specific MPs.
The second optimization approach was directed at the CF lysate itself. CF synthesis for NMR analysis benefits from selective labeling schemes enabled by truncated amino acid (AA) metabolic pathways in lysates, reducing spectral ambiguity. However, residual enzymatic AA conversions persist, leading to label dilution and ambiguous NMR spectra. This study aimed to eliminate these residual activities in the E. coli A19 strain, generating optimized CF lysates for NMR applications.
The approach involved cumulative gene deletions of the most problematic scrambling enzymes. The new strain, “Stablelabel,” included deletions and modifications in genes asnA, ansA, ansB, glnA, aspC, and ilvE, effectively eliminating background activities of L-Asn, L-Asp, and conversions of L-Glu to L-Asp and L-Gln. However, residual conversion of L-Gln to L-Glu persisted due to glutaminase activity of several glutaminases using the inhibitor 6 diazo-5-oxo-L-norleucine (DON). Stablelabel showed a slightly slower growth than A19, and an overall good performance with 2.7 mg/mL GFP expressed in the reaction mixture (RM) compared to the parental A19 strain with 3.5 mg/mL. Furthermore, the strain was successfully applied to demonstrate methyl group labeling of MPs using preconverted L-val and L-leu from their respective precursors 2-ketoisovalerate and 4-methyl-2-oxovalerate.
In this study, lipid nanoparticle particle-and strain engineering vividly demonstrated the potential of CFPS systems and their versatility. While the SapNP system requires further engineering to potentially reach the efficiency of the well-studied MSP NDs, this study provides an example of nanoparticle characterization allowing new insights into NP behavior in CF systems. Furthermore, it was shown that strain engineering is a straightforward solution to tailor CF lysates to the individual requirements. After this thesis was submitted, Stablelabel in fact was successfully applied for backbone assignment of casein kinase 1, thereby demonstrating its suitability to express complex targets for NMR studies.
Der retinoid-related orphan receptor α (RORα) ist ein nukleärer Rezeptor, der nach Bindung an sein Responselement die Transkription zahlreicher Gene reguliert. Pharmazeutisches Interesse erlangt der Rezeptor vor allem durch seine Verwicklung in pathophysiologische Prozesse wie Osteoporose und Arteriosklerose sowie durch seine antiinflammatorische Wirkung, die auf der negativen Interferenz mit dem NF-κB-Signalweg beruht. Bisher konnten vier RORα-Isoformen isoliert werden, die durch alternatives Spleißen sowie durch die Regulation über unterschiedliche Promotorregionen entstehen. In verschiedenen Studien konnte eine isoformspezifische Regulation als Antwort auf pathophysiologische Veränderungen der Zellen festgestellt werden, wie beispielsweise die Induktion der RORα4-Transkription in Leberzellen infolge einer Sauerstoffunterversorgung. Um Einblicke in die Mechanismen zu gewinnen, die der spezifischen Regulation der RORα4-Expression zugrunde liegen, wurde in der vorliegenden Arbeit der RORα4-Promotor als erster Promotor einer RORα-Isoform identifiziert und analysiert.
Sechs Fragmente mit einer Länge von bis zu 5,1 kbp der aus Datenbanken entnommenen, putativen Promotorsequenz wurden in einen Reportergenvektor kloniert. Transiente Transfektionsexperimente und Reportergenanalysen deckten die Promotoraktivität der gewählten Sequenz auf.
In dem durch einen hohen Gehalt an den Nukleotiden G und C auffallenden Promotor wurden drei einzelne GC-Boxen (A, B und C) sowie eine Viererkette (Box D) und eine Tandem-GCBox (Box E) als mögliche Bindungsmotive für Sp-Transkriptionsfaktoren gefunden. Mithilfe von Kotransfektionen konnte eine Induktion der Promotoraktivität durch die Transkriptionsfaktoren Sp1 und Sp4 nachgewiesen werden, während Sp3 die Promotoraktivität in diesen Experimenten nicht beeinflusste.
Durch die gezielte Mutation oder Deletion, bzw. die Inkubation mit verschiedenen Substanzen konnten diesen GC-Boxen unterschiedliche Funktionen zugeordnet werden. Durch transiente Transfektionen stark verkürzter Promotorfragmente wurde ein für die Promotoraktivität nötiger Sequenzbereich von 170 Basenpaaren eingegrenzt. In Mutationsanalysen wurde demonstriert, dass die beiden proximalen GC-Boxen A und B für die basale Promotoraktivität essentiell sind.
Die RORα4-Promotoraktivität ließ sich zelltypabhängig durch den Phorbolester TPA induzieren. In Deletionsanalysen ließ sich dieser Effekt teilweise auf die GC-Boxen C und D zurückführen. Der distalen GC-Box E konnte ebenfalls eine Funktion zugeordnet werden. In Reportergenanalysen konnte demonstriert werden, dass sie die Induktion der Promotoraktivität durch den HDAC-Inhibitor Trichostatin A vermittelt.
Durch die Untersuchungen an den TK-luc-Konstrukten mit RORα-Responselementen konnte gezeigt werden, dass der virale Promotor aufgrund der einklonierten RORα-Responselemente sehr stark auf die Kotransfektion der RORα-Isoformen reagiert. Die Reportergenanalyse mit diesen Konstrukten stellt daher eine effiziente Methode dar, um die RORα-vermittelte Transaktivierung zu bestimmen.
Obwohl der RORα4-Promotor zahlreiche RORα-Responselemente trägt, konnte in den Kotransfektionen mit Expressionsplasmiden für die einzelnen Isoformen in keiner der drei Zelllinien eine Autoregulation gefunden werden. Ebensowenig zeigte sich ein Einfluss des putativen RORα-Liganden Melatonin auf die Promotoraktivität.
Des Weiteren wurde gezeigt, dass die RORα4-Promotoraktivität in HeLa und MCF-7-Zellen durch das cAMP-Analogon DbcAMP induzierbar ist, während in HEK 293 keine Beeinflussung der Promotoraktivität erzielt wurde. Neben der Steigerung der Promotoraktivität durch TPA, konnte mit der DbcAMP-Induktion folglich ein zweiter, zelltypabhängiger Effekt auf die RORα4-Promotoraktivität identifiziert werden.
The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death
(2023)
Every day, living organisms are challenged by internal and external factors that threaten to bring imbalance to their tightly regulated systems and disrupt homeostasis, leading to degeneration, and ultimately death. More than ever, we face the challenge of combating diseases such as COVID-19 caused by infection with the SARS-CoV-2 coronavirus. It is therefore crucial to identify host factors that control antiviral defense mechanisms. In addition, in the fight against cancer, it is becoming increasingly important to identify markers that could be used for targeted therapy to influence cellular processes and determine cell fate.
As a deubiquitylating enzyme, ubiquitin specific peptidase 22 (USP22) mediates the removal of the small molecule ubiquitin, which is post-translationally added to target proteins, thereby regulating several important processes such as protein degradation, activation or localization. Through its deubiquitylating function, USP22 controls several biological processes such as cell cycle regulation, proliferation and cancer immunoresistance by modulating key proteins involved in these pathways. Lately, USP22 was reported to positively regulate TNFα-mediated necroptosis, an inflammatory type of programmed cell death, in various human tumor cell lines by affecting RIPK3 phosphorylation. In addition, USP22 as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription complex is known to regulate gene expression by removing ubiquitin from histones H2A and H2B. However, little is known about the role of USP22 in global gene expression.
In this study, we performed a genome-wide screen in the human colon carcinoma cell line HT-29 and identified USP22 as a key negative regulator of basal interferon (IFN) expression. We further demonstrated that the absence of USP22 results in increased STING activity and ubiquitylation, both basally and in response to stimulation with the STING agonist 2'3'-cGAMP, thereby affecting IFNλ1 expression and basal expression of antiviral ISGs. In addition, we were able to establish USP22 as a critical host factor in controlling SARS-CoV-2 infection by regulating infection, replication, and the generation of infectious virus particles, which we attribute in part to its role in regulating STING signaling.
In the second part of the study, we connected the findings of USP22-dependent regulation of IFN signaling and TNFα-induced necroptosis and investigated the role of USP22 during necroptosis induced by the synergistic action of IFN and the Smac mimetic BV6 in caspase-deficient settings. We identified USP22 as a negative regulator of IFN-induced necroptosis, which does not depend on STING expression, but relies on a yet unknown mechanism.
In summary, we identify USP22 as an important regulator of IFN signaling with important implications for the defense against viral infections and regulation of the necroptotic pathway that could be exploited for devising targeted therapeutic strategies against viral infections and related diseases like COVID-19, and advancing precision medicine in cancer treatment.
Necroptosis is an immunogenic form of programmed cell death characterized by plasma membrane accumulation of activated mixed lineage kinase domain-like (MLKL) that eventually leads to membrane disruption and release of danger-associated molecular patterns (DAMPs). Necroptotic cell death is tightly controlled by checkpoints, including compartmentalization as well as post-translational modifications (PTMs), like phosphorylation and ubiquitination of receptor-interacting protein kinase (RIPK) 1, RIPK3 and MLKL. Removal of plasma membrane-located activated MLKL via endocytosis or exocytosis can counteract necroptosis, but up till now, the exact mechanisms by which necroptosis is regulated downstream of MLKL activation and oligomerization are not fully understood.
Ubiquitination is a key post-translational modification that regulates various cellular processes including cell survival and cell death signaling via ubiquitination of RIPK1, RIPK3 and MLKL. M1-linked (linear) poly-ubiquitination is mediated exclusively by the linear ubiquitin chain assembly complex (LUBAC) which critically regulates cell fate and immune signaling via death receptors such as TNF receptor 1 (TNFR1).
In this study, we demonstrate that M1 poly-Ubiquitin (poly-Ub) increases during necroptosis which can be blocked by inhibition of LUBAC activity with the small-molecule HOIL-1-interacting protein (HOIP) inhibitor HOIPIN-8 or by loss of LUBAC catalytic subunit HOIP. Intriguingly, HOIPIN-8, as well as the HOIP inhibitor gliotoxin, and HOIP knockdown effectively prevent TNFα/smac mimetic/zVAD.fmk-induced necroptotic cell death in cells of human origin, without affecting necroptotic RIPK1 and RIPK3 phosphorylation, necrosome formation and oligomerization of phosphorylated MLKL. We demonstrate that HOIPIN-8 treatment inhibits MLKL translocation to intracellular membranes and accumulation in plasma membrane hotspots as well as MLKL exocytosis. We further confirm that HOIPIN-8 treatment suppresses necroptotic cell death in primary human pancreatic organoids (hPOs). Using time-lapse imaging and live/dead staining, we demonstrate loss of organoid structure and hPO cell death induced by smac mimetics and caspase inhibitors, thus providing a novel platform to investigate necroptosis in near physiological settings. Inhibition of LUBAC activity with HOIPIN-8 prevents hPO collapse and extends cell viability. Of note, loss of the M1 Ub-targeting deubiquitinating enzymes (DUBs) OTU DUB with linear linkage specificity (OTULIN) and cylindromatosis (CYLD) in human cell lines does not affect necroptosis induction and HOIPIN-8-mediated rescue of necroptosis. Intriguingly, inhibition of LUBAC activity with HOIPIN-8 does not block necroptotic cell death in murine cell lines.
Using massive analyses of cDNA ends (MACE)-seq-based global transcriptome analysis we confirm that necroptosis induces a pro-inflammatory cytokine profile which is dependent on LUBAC function and necroptotic signaling. Loss of LUBAC activity prevents the MLKL-dependent production and release of pro-inflammatory cytokines and chemokines.
Finally, we identify Flotillin-1 and -2 (FLOT1/2) as putative targets of necroptosis-induced M1 poly-Ub. Ubiquitin-binding in ABIN and NEMO (UBAN)-based pulldowns of M1 poly-ubiquitinated proteins revealed enrichment of FLOTs after necroptosis induction which is dependent on LUBAC activity and can be blocked with necroptosis inhibitors Nec-1s, GSK’872 and NSA, targeting RIPK1, RIPK3 and MLKL, respectively. Of note, loss of FLOT1/2 potentiates necroptosis suppression induced by LUBAC inhibition with HOIPIN-8.
Together, these findings identify LUBAC-mediated M1 poly-Ub as an important mediator of necroptosis and identify FLOTs as novel putative targets of LUBAC-mediated M1 poly-Ub during necroptosis. In addition, by modeling necroptosis in primary human organoids, we further expand the spectrum of experimental models to study necroptosis in human cellular settings.
Mitochondria are important for cellular health and their dysfunction is linked to a variety of diseases, especially neurodegeneration. Thus, the renewal and degradation of dysfunctional mitochondria is crucial for the well-being of organisms. The selective digestion of damaged mitochondria via the lysosome (mitophagy), is the main pathway to do so.
In my dissertational work, I investigated the connection between protein misfolding, protein import into mitochondria and the degradation of mitochondria via mitophagy. Here, I present a new model for the initiation of mitophagy without collapse of the membrane potential. This model provides the link between protein import into mitochondria, stress signal transduction to the cytosol and the mitochondrial stress sensor PINK1. To comprehensively examine how mitophagy can be triggered, I performed a genome-wide CRISPR knockout screen utilizing the mitophagy reporter mitochondrial mKEIMA. Thereby, I observed numerous novel gene deletions that induce mitophagy. Prominently, I identified an accumulation of gene deletions of the protein import and of protein quality control factors. I validated several of those and examined HSPA9 (mitochondrial HSP70) and LONP1 (a mitochondrial matrix AAA protease) in more detail, regarding their effect on mitophagy and protein import. For this, I used an established fluorescence-based, mitochondrial-targeted EGFP, as well as a newly-developed pulsed-SILAC mass spectrometry approach (mePRODmt). Depletions of both genes resulted in reduced protein import and PINK1-dependent mitophagy. Strikingly, I did not observe any loss of mitochondrial membrane potential, which was hitherto believed to be essential for activation of PINK1-mediated mitophagy. Literature shows that certain mitochondrial stressors can also induce mitophagy without mitochondrial membrane depolarization, which I confirmed with my assays. Next, I characterized the impact of LONP1 and HSPA9 depletion, which are involved in proteostasis maintenance, and the mtHSP90 inhibitor GTPP on mitochondrial protein folding in more detail. GTPP treatment and LONP1 depletion both resulted in the accumulation of an insoluble protein fraction, as judged by proteomic analysis. This insoluble protein fraction enriched several components of the presequence translocase-associated motor PAM, including TIMM44. TIMM44 acts as a link between the translocon, the import pore of the inner mitochondrial membrane (TIM) complex and the PAM complex. Thus, I hypothesized that TIMM44 dissociates from the TIM complex upon protein folding stress, when it becomes part of the insoluble protein fraction. To validate this model, I measured the TIMM44 interactome upon proteostasis disturbance using proximity labeling. Indeed, interaction of TIMM44 with the import pore was almost completely abolished, explaining the loss of matrix-targeted import upon protein folding stress. From these findings, I reasoned that an import reduction mediated by the PAM complex would likely also inhibit the degradation of PINK1. Consistent with this hypothesis, I observed that mitophagy induced by HSPA9 or LONP1 deletion was prevented when PINK1 was genetically deleted. In comparison, non-processed PINK1 was stabilized on mitochondria in wild type cells when mitochondrial protein import was impaired. On this basis, I drew the conclusion that the loss of mitochondrial import was the stress signal, which leads to the stabilization of PINK1, as it could not be processed anymore via the inner mitochondrial membrane protease PARL. PINK1 auto-activates itself upon accumulation and signals to the cytosol that this mitochondrion is damaged. Mitophagy is subsequently initiated by the ubiquitin kinase activity of PINK1. As a result, the autophagy apparatus gets activated, damaged mitochondria are engulfed by a double membrane and removed via lysosomal digestion. This proposed model is, to the best of my knowledge, the first to provide an explanation for protein folding stress-induced and protein import inhibition-triggered mitophagy without mitochondrial depolarization. The model thus extends the PINK1/PARKIN-dependent mitophagy pathway to milder stresses and clears some of the open questions in the field. Furthermore, this work is also important, because protein misfolding stress and dysfunctional mitochondria are two hallmarks of neurodegeneration. In particular, mitochondrial protein import inhibition during Parkinson’s and Huntington disease might be driver of mitochondrial dysfunction. Hence, I hope and anticipate that the newly developed protein import method, mePRODmt, and the proposed model will be beneficial to further characterize underlying processes and to establish which factors prevent or drive these disorders on molecular level.
Lysosomes are major degradative organelles that contain enzymes capable of breaking down proteins, nucleic acids, carbohydrates, and lipids. In the last decade, new discoveries have traced also important roles for lysosomes as signalling hubs, affecting metabolism, autophagy and pathogenic infections. Therefore, maintenance of a healthy lysosome population is of utmost importance to the cell to respond to both stress conditions and also homeostatic signalling. For example, for minor perturbations to the lysosomal membrane, the cell activates repair processes which seal membrane nicks. For more extensive damage, autophagy is activated to remove damaged organelles from the cell. on the other hand, during pathogen invasion host cells have also evolved mechanisms to hijack the endolysosomal pathway to facilitate their own growth and replication in host cells.
The first part of the thesis work focuses on a lysosomal regeneration program which is activated under conditions where the entire lysosomal pool of the cell is damaged. Upon extensive membrane damage induced by the lysosomotropic drug LLOMe, the cell activates a regeneration pathway which helps in the formation of new functional lysosomes by recycling damaged membranes. I have identified the molecules important for this novel pathway of lysosomal regeneration and showed how the protein TBC1D15 orchestrates this process to regenerate functional organelles from completely damaged membrane masses in the first 2 hours following lysosomal membrane damage. This process resembles the process of auto- lysosomal reformation (ALR)- involving the formation of lysosomal tubules which are extended along microtubules and cleaved in a dynamin2 dependent manner to form proto-lysosomes which develop into fully functional mature lysosomes. These lysosomal tubules are closely associated with ATG8 positive autophagosomal membranes and require ATG8 proteins to bind to the lysophagy receptor LIMP2 on damaged membranes. This process is physiologically important under conditions of crystal nephropathy where calcium oxalate crystals induce damage to lysosomal membranes in nephrons in kidney disease.
The second part of the thesis shows how the endolysosomal system of the cell is hijacked by the bacteriaLegionella pneumophila. During Legionella infection the formation of conventional ATG8 positive autophagosomes are blocked due to the protease activity of the bacterial effector protein RavZ which cleaves lipidated ATG8 proteins from autophagosomal membranes. The SidE effectors of Legionella modify STX17 and SNAP29 by the process of non-canonical ubiquitination called phosphoribose-linked serine ubiquitination (PR-Ub). These proteins are essential for the formation of the autophagosomal SNARE complex which is used for fusion of the autophagosome with the lysosome. Upon Legionella infection, PR-UB of STX17 aids in formation of autophagosome-like replication vacuoles. ThesevacuolesdonotfusewiththelysosomebecauseSNAP29isalsoPR-Ubmodified. PR-UbofSTX17 and SNAP29 sterically blocks the formation of the autophagosomal-SNARE complex thereby preventing fusion of the autophagosome with the lysosome. As a result, Legionella can replicate in autophagosome- like vacuoles which do not undergo lysosomal degradation. In absence of PR-Ub modified STX17, bacterial replication is compromised when measured by bacterial replication assays in lung epithelial (A549) cells.
Taken together, this thesis highlights two important aspects of the autophagy-lysosomal system- how it responds to extensive membrane damage and its importance in Legionella pneumophila infection. Extensive damage to lysosomal membranes triggers a rapid regeneration process to partially restore lysosomal function before the effects of TFEB dependent lysosomal biogenesis becomes apparent. On the other hand, Legionella pneumophila infection segregates the lysosomes from the rest of the endo-lysosomal system by blocking autophagosome-lysosome fusion. Though lysosomes remain active, they are incapable of degrading pathogens since pathogen containing vacuoles do not fuse with the lysosome.
Membrane proteins are a diverse group of proteins that serve a multitude of purposes with one of the most important ones being transport. All kinds of substrates are shuffled over biological membranes with the help of dedicated proteins enabling the transport along and against a concentration gradient. Within the group of actively transporting proteins a diverse set of proteins that rely on an electrochemical gradient to facilitate transport of a substrate against its concentration gradient can be found. Those so-called secondary active
transporters are a group on integral membrane proteins ubiquitous to all cells. They allow the transport of all kinds of substrates like nutrients, ions, other metabolites and drugs over the hydrophobic barrier created by the cellular and organellar membrane. The gradients that provide the main driving force for most of the transporters are either sodium ions or protons, although transporters utilizing other ions or organic compounds are found as well. In case of exchangers two very similar substrates are transported in opposing direction over the membrane, one against its electrochemical gradient driven by the other.
Along with a structural diversity of the transporters concerning overall shape, oligomerization and number of transmembrane elements comes a mechanistic variety though still following the principle of alternating access. In humans the malfunction of secondary active transporters can lead to a physiological disorders such as epilepsy, depression or obesity.
The focus of this thesis was the structural and functional characterization of the secondary active transporter SeCitS from Salmonella enterica, a symporter of the 2-hydroxycarboxylate family. The transport of citrate as a bivalent ion is facilitated by the flux of sodium ions that have an inward-facing gradient over the inner membrane of Salmonella enterica. Transport experiments showed that the transport ratio is two sodium ions per citrate molecule, netting in an electroneutral transport. Compared to other members of the family the specificity of the transporter towards its main substrate is very high.
Structural information on the protein was initially obtained through 2D electron crystallography, which allowed the identification of the oval shaped dimer and a first hint towards a significant conformational change that the protein undergoes during its transport cycle. Using 3D crystallography, the X-ray structure of the transporter was solved. The protein crystalizes as a stable, but conformationally asymmetric dimer. As bound citrate can be readily identified in both protomers they can be assigned into an outward- and an inward-facing conformation, with the main citrate binding site in the outward-facing conformation.
One interesting feature of the crystal structure was the large surface available for multimerization, providing a platform for tight dimerization of the two protomers. On the other hand, SeCitS did not show a true cooperativity of transport. With those two aspects taken into account the question arose if any potential crosstalk between the monomers within the dimer takes place and influences transport (negative cooperativity) or the conformational distribution within the dimer (stabilization of the protein within the membrane).
The functional approach in answering this question was the use of mutated variants of the protein for cross-linking within one monomer. Two residues were chosen respectively to lock one of either conformation to be able to test for transport activity in the remaining protomer. The suitability of the residues was derived from the crystal structure (D112 – R205 to lock the inward-facing conformation and L337 – S412 for the outward-facing conformation). After initial promising results the final variants were not stable enough to be analyzed in transport assays.
To analyze the distribution of relative conformations within the dimer the protein was reconstituted into native-like lipid environment such as nanodiscs or saposin nanoparticles to be analyzed by cryo-electron microscopy. The first images were recorded and did yield promising 2D classes where the general features of the transporter were identified. Yet, an improved preparation is required to obtain a high resolution structure.
The key functional aspects of a transporter are its ability to bind and transport its substrates. In a set of experiments those features were investigated by a radioligand transport assay and by isothermal titration calorimetry (ITC). The transport properties of the protein were assessed in a filter assay using a radioactively labeled citrate as a read-out. The protein was reconstituted into proteoliposomes and subjected to different substrate conditions. Different ions were tested in its ability to drive or inhibit transport, but only sodium ions were able to drive transport and also not hindered by the presence of other ions...
Caspase-2 is the evolutionary most conserved member of the caspase family and was shown to be involved in genotoxic stress induced apoptosis, control of aneuploidy, and ageing related metabolic changes. However, its role in apoptosis seems redundant due to the observation, that knockout does not inhibit apoptotic signalling exclusively. Instead, knockout of caspase-2 leads to tumor susceptibility in vivo, which led to the assumption, that caspase-2 has non-apoptotic functions and can act as a tumor suppressor. The underlying mechanism of the tumor suppressor activity of caspase-2 has not been clarified so far. Furthermore, caspase-2, has a prominent, and as pro-enzyme exclusive localisation in the nucleus and other subcellular compartments, implicating a distinct and location specific role.
In this study, a novel caspase-2 specific substrate, termed p54nrb, was identified. P54nrb is harbouring a caspase-2 specific cleavage site at the aspartate residue D422, and cleavage of p54nrb leads apparently to disruption of its putative DNA binding domain at the C-terminus.
P54nrb is a nuclear multifunctional RNA and DNA binding protein, known for roles in transcriptional regulation, DNA unwinding and repair, RNA splicing, and retention of defective RNA. Overexpression of p54nrb has been observed in several human cancers, such as cervix carcinoma, melanoma, and colon carcinoma.
Data from this study revealed, that depletion of p54nrb in tumor cell lines results in a loss of resistance to drug induced cell death and to reduced capability of anchorage independent growth, which is functionally equivalent to a reduced tumorigenic potential. Meanwhile, p54nrb depletion alone is not cytotoxic.
The investigation of p54nrb dependent gene regulations by high resolution quantitative proteomics uncovered an altering expression of multiple tumorigenic genes. For two of these candidates, the tumorigenic protease cathepsin-Z and the anti-apoptotic gelsolin, p54nrb dependent expression was detected universally in all three investigated tumor cell lines, cervix carcinoma, melanoma, and colon carcinoma. Additionally, a direct interaction of p54nrb with the cathepsin Z and gelsolin encoding DNA, but not with their corresponding mRNA, could be demonstrated.
Conjointly, this study unveils a novel mechanistic feature of caspase-2 as a tumor suppressor. The caspase-2—p54nrb axis can orchestrate the levels of several tumorigenic proteins and thereby determine the cell death susceptibility and long-term tumor survival. These findings might be of great value for future therapeutic interventions and for overcoming drug resistance of tumors.