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The absolute configuration of the title molecule, [Fe(C5H5)(C38H34NP2)]·CHCl3, is R,Rp. The molecular structure is similar to the structure of the solvent-free compound [Fukuzawa, Yamamoto & Kikuchi (2007). J. Org. Chem. 72, 1514-1517], but some torsion angles about the P-Cphenyl bonds differ by up to 25°. The P atoms and the N atom have a distorted trigonal-pyramidal geometry. The chloroform solvate group donates a C-H...[pi] bond to the central benzene ring and is also involved in six intermolecular C-H...Cl contacts with H...Cl distances between 2.96 and 3.13 Å. Key indicators: single-crystal X-ray study; T = 163 K; mean σ(C–C) = 0.003 Å; R factor = 0.039; wR factor = 0.088; data-to-parameter ratio = 24.2.
The title molecule, C34H28I4·4C6H6, has crystallographic 4 symmetry and crystallizes with four symmetry-related benzene solvent molecules. The phenyl group is eclipsed with one of the adamantane C—C bonds. The tetraphenyladamantane units and the benzene solvent molecules are connected by weak intermolecular phenyl–benzene C—H⋯π and benzene–benzene C—H⋯π interactions. In the crystal, molecules are linked along the c-axis direction via the iodophenyl groups by a combination of weak intermolecular I⋯I [3.944 (1) Å] and I⋯π(phenyl) [3.608 (6) and 3.692 (5) Å] interactions.
Dichlorido(3-phenylindenylidene)bis(triphenylphosphane)ruthenium(II) tetrahydrofuran disolvate
(2011)
The RuII atom in the title compound, [RuCl2(C15H10)(C18H15P)2]·2C4H8O, has a distorted square-pyramidal conformation. The P and Cl atoms are at the base of the pyramid and the Ru-Cindenylidene bond is in the axial position. The two Cl ligands and the two phosphane ligands are in trans positions. The Cl-Ru-Cl and P-Ru-P angles are 157.71 (2) and 166.83 (2)°, respectively. The two independent tetrahydrofuran (THF) solvent molecules are disordered. One THF molecule was refined using a split-atom model. The second THF molecule was accounted for by using program PLATON/SQUEEZE [Spek (2009). Acta Cryst. D65, 148-155]. The molecular conformation shows three intramolecular C-H...Cl contacts and two C-H...[pi] interactions while the crystal packing features an intermolecular C-H...Cl contact and two very weak intermolecular C-H...[pi] contacts.
The six-membered ring of the title compound, C11H16NO, has a distorted envelope conformation. The piperidine N atom deviates by 0.128 (1) Å from the plane through its three neighbouring atoms. In the crystal structure, molecules are connected by intermolecular Cethynyl-H...O contacts to form chains extending in the [10\overline{1}] direction. Key indicators: single-crystal X-ray study; T = 167 K; mean σ(C–C) = 0.001 Å ; R factor = 0.040; wR factor = 0.112; data-to-parameter ratio = 27.3.
The absolute configuration of the title compound, [Fe(C5H5)(C36H29OP2)], is Sp at the ferrocene group and S at the asymmetric C atom. Both P atoms have a trigonal-pyramidal conformation. There is a short intramolecular C-H...P contact with an H...P distance of 2.56 Å. The hydroxy group is involved in an intramolecular O-H...[pi]phenyl interaction. The crystal packing shows five very weak intermolecular C-H...[pi] contacts, with H...Cg distances between 3.26 and 3.39 Å (Cg is the centroid of a phenyl or cyclopentadienyl ring). Key indicators: single-crystal X-ray study; T = 162 K; mean σ(C–C) = 0.004 Å; R factor = 0.038; wR factor = 0.083; data-to-parameter ratio = 22.3.
Antibiotic resistance of pathogenic bacteria is a major worldwide problem. Bacteria can resist antibiotics by active efflux due to multidrug efflux pumps. The focus of this study has been the mycobacterial multidrug transporter TBsmr because it belongs to the small multidrug resistance (SMR) family whose members are a paradigm to study multidrug efflux due to their small size. SMR proteins are typically 11-12 kDa in size and have a four-transmembrane helix topology. They bind cationic, lipophilic antibiotics such as ethidium bromide (EtBr) and TPP+, and transport them across the membrane in exchange for protons. To understand the molecular mechanism of multidrug resistance, we have to gain information about the structure and function of these proteins. The research described in this thesis aimed to deduce details about the topology, transport cycle and key residues of TBsmr using biophysical techniques. Solid-state NMR (ssNMR) can provide detailed insight into structural organization and dynamical properties of these systems. However, a major bottleneck is the preparation of mg amounts of isotope labeled protein. In case of proteoliposomes, the problem is compounded by the presence of lipids which have to fit into the small active volume of the ssNMR rotor. In Chapter 3, an enhanced protein preparation is described which yields large amounts of TBsmr reconstituted in a native lipid environment suitable for further functional and structual studies. The achieved high protein-to-lipid ratios made a further characterization by ssNMR feasible. The transport activity and oligomeric state of the reconstituted protein in different types of lipid was studied as shown in Chapter 4. The exact oligomeric state of native SMR proteins is still uncertain but a number of biochemical and biophysical studies in detergent suggest that the minimal functional unit capable of binding substrate is a dimer. However, binding assays are not ideal since a protein may bind substrate without completing the transport cycle which can only be shown for reconstituted protein in transport assays.By combining functional data of a TPP+ transport assay with information about theoligomeric state of reconstituted TBsmr obtained by freeze-fracture electron microscopy, it could be shown that lipids affect the function and the oligomeric state of the protein, and that the TBsmr dimer is the minimal functional unit necessary for transport. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. In Chapter 5, the existence of such a substrate-transporter complex for the TBsmr and its substrate EtBr could be shown. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. The measurements have shown the formation of a pH-dependant, transient substrate-protein complex between binding and release of EtBr. This state was further characterized by determining the Kd, by inhibiting EtBr transport through titration with non-fluorescent substrate and by fluorescence anisotropy measurements. The findings support a model with a single occluded intermediate state in which the substrate is highly immobile. Liquid-state NMR is a useful tool to monitor protein-ligand interactions by chemical shift mapping and thus identify and characterize important residues in the protein which are involved in substrate binding. In agreement with previous studies (Krueger-Koplin et al., 2004), the detergent LPPG was found to be highly suitable for liquid-state NMR studies of the membrane protein TBsmr and 42% of the residues could be assigned, as reported in Chapter 6. However, no specific interactions with EtBr were found. This observation was confirmed by LILBID mass spectrometry which showed that TBsmr was predominantly in the non-functional monomeric state. Functional protein was prepared in proteoliposomes which can be investigated by solidstate NMR (Chapter 7). Besides the essential E13, the aromatic residues W63, Y40, and Y60 have been shown to be directly involved in drug binding and transport. Different isotope labeling strategies were evaluated to improve the quality of the NMR spectra to identify and characterize these key residues. In a single tryptophan mutant of reconstituted TBsmr W30A, the binding of ethidium bromide could be detected by 13C solid-state NMR. The measurements have revealed two populations of the conserved W63 residue with distinct backbone structures in the presence of substrate. There is a controversy about the parallel or anti-parallel arrangement of the protomers in the EmrE dimer (Schuldiner, 2007) but this structural asymmetry is consistent with both a parallel and anti-parallel topology.
Autophagy, together with the ubiquitin-proteasome system, is the main quality control pathway responsible for maintaining cell homeostasis. There are several types of autophagy distinguished by cargo selectivity and means of induction. This thesis focuses on macroautophagy, hereafter autophagy, where a double-layered membrane is formed originating from the endoplasmatic reticulum (ER) engulfing cargo selectively or unselectively. Subsequently, a vesicle forms around the cargo, an autophagosome, and eventually fuses with the lysosome leading to degradation of the vesicle content and release of the cargo “building blocks”. Basal autophagy continuously occurs, unselectively engulfing a portion of the cytoplasm. However, autophagy can also be induced by stress such as starvation, protein aggregation, damaged organelles, intracellular pathogens etc. In this case, the cargo is selectively targeted, and the fate of the autophagosome is the same as in basal autophagy. In recent years, interest in identifying mechanisms of autophagy regulation has risen due to its importance in neurodegenerative diseases and cancer. Given the complexity of the process, its execution is tightly regulated from initiation, autophagosome formation, expansion, closure, and finally fusion with the lysosome. Each of the steps involves different protein complexes, whose timely activity is orchestrated by post-translational modifications. One of them is ubiquitination. Ubiquitin is a small, 76-amino acid protein conjugated in a 3-step reaction to other proteins, in a reversible manner, meaning undone by deubiquitinases. Originally described as a degradation signal targeting proteins to the proteasome, today it is known it has various additional non-proteolytic functions, such as regulating a protein’s activity, localization, or interaction partners. The role of ubiquitin in autophagy has already been shown. However, given the reversibility and fine-tuning of the ubiquitin signal, many expected regulators remain unidentified. This work aimed to identify novel deubiquitinating enzymes that regulate autophagy. We identified ubiquitin-specific protease 11 (USP11) as a novel, negative regulator of autophagy. Loss of USP11 leads to an increase in autophagic flux, whereas overexpression of USP11 attenuates it. Moreover, this observation was reproducible in model organism Caenorhabditis elegans, emphasizing the importance of USP11 in autophagy regulation. To identify the mechanism of USP11-dependent autophagy regulation, we performed a USP11 interactome screen after 4 hour Torin1 treatment and identified a plethora of autophagy-related proteins. Following the most prominent hits, we have investigated versatile ways in which USP11 regulates autophagy. USP11 interacts with the PI3KC3 complex, the role of which is phosphorylating lipids of the ER, thereby initiating the formation of the autophagosomal membrane. Phosphorylated lipids serve as a recruitment signal for downstream effector proteins necessary for the membrane expansion. The core components of the complex are VPS34, the lipid kinase, ATG14, the protein responsible for targeting the complex to the ER, VPS15, a pseudokinase with a scaffolding role, Beclin1, a regulatory subunit, and NRBF2, the dimer-inducing subunit. We have found USP11 interacts with the complex and, based on its activity, USP11 influences post-translational status of all the aforementioned subunits, except for ATG14. Moreover, we have found that loss of USP11 leads to an increase in NRBF2 levels, whereas it does not change the levels of the other proteins. Given that the dimerization of the complex leads to an increase in complex activity, we investigated if the complex is more tightly formed in the absence of USP11, and if it is more active. We have found both to be the case. Although the exact mechanism of USP11-dependent PI3KC3 complex regulation remains to be identified, we found that loss of USP11 stimulates the complex formation and activity, likely contributing to the general effect of USP11 on autophagy flux. Additionally, we found that USP11 modulates levels of mTOR, the most upstream kinase in autophagy initiation steps and general multifaceted metabolism regulator. Loss of USP11 led to downregulation of mTOR levels, suggesting USP11 may rescue mTOR from proteasome-mediated degradation. Furthermore, we found mTOR to be differentially modified depending on the activity of USP11. However, it remains to be shown if USP11-dependent mTOR regulation contributes to the observed autophagy phenotype. Taken together, USP11 is a novel, versatile, negative regulator of autophagy, and an important addition to our knowledge on the regulation of autophagy by the ubiquitin system.
5-Lipoxygenase (5LO) is a key enzyme in biosynthesis of leukotrienes (LTs), lipid mediators of inflammation. To study the roles of the 5LO accessory proteins coactosin-like protein (CLP) and 5LO-activating protein (FLAP), we knocked down these proteins in human monocytic cells. Our results show that expression of CLP was required for full cellular 5LO activity when cells were activated with Ca2+ ionophore, as well as with a physiological stimulus (lipopolysaccharide followed by N-formylmethionyl-leucyl-phenylalanine). During LT biosynthesis in stimulated cells, 5LO typically translocates to the nuclear membrane. This redistribution, from cytosolic to perinuclear, was clearly compromised in both CLP- and FLAP-deficient cells. Our results suggest that the CLP–5LO interaction may be a target for reduced LT production.
In der vorliegenden Doktorarbeit wurden die folgenden fünf biochemischen Fragestellungen zu Enzymen und deren Wechselwirkungen mit ihren Substraten mit NMR spektroskopischen Methoden untersucht: (1) Die spontane Bildungsrate von NCarboxymethanofuran (Carbamat) aus CO 2 und Methanofuran im Gleichgewicht und die Beeinflussung der Geschwindigkeit durch FormylmethanofuranDehydrogenase aus Methanosarcina barkeri wurden über 2D ProtonenaustauschSpektroskopie (EXSY) bestimmt. Mit Hilfe der berechneten Geschwindigkeitskonstanten und der bekannten physiologischen Konzentrationen von CO 2 und Methanofuran konnte erstmals gezeigt werden, daß die spontane Carbamatbildungsrate ausreicht, um die in vivoCO 2 Reduktionsraten in methanogenen Archaea erklären zu können. (2) Die Konformation von N 5 ,N 10 Methylentetrahydromethanopterin (Methylen H 4 MPT) in Anwesenheit und in Abwesenheit H 2 bildender MethylenH 4 MPTDehydrogenase aus Methanothermobacter marburgensis wurde über TransferNOEMessungen bestimmt. Es wurde nachgewiesen, daß die Bindung zu einer Konformationsänderung des Substrats führt, welche die ReSeitenStereospezifität des Enzyms erklären kann. (3) Die Stereospezifität des Hydridtransfers von MethylenH 4 MPT auf NADP , katalysiert von NADPabhängiger MethylenH 4 MPTDehydrogenase aus Methylobacterium extorquens AM1, wurde NMRspektroskopisch untersucht. In Übereinstimmung mit der unter (2) entwickelten Theorie zum Übergangszustand des Hydridtranfers wurde gefunden, daß auch der Transfer auf NADP ReSeitenspezifisch in Bezug auf MethylenH 4 MPT verläuft. Zusätzlich wurde gezeigt, daß das Enzym das Hydrid auf die ReSeite von NADPH überträgt. (4) Die spontane Rate der Kondensationsreaktion von Glutathion und Formaldehyd zu SHydroxymethylglutathion wurde mit EXSYMessungen bestimmt. In Zellextrakten des methylotrophen Proteobakteriums Paracoccus denitrificans wurde eine Enzymaktivität nachgewiesen, die diese Reaktion beschleunigt. Bislang wurde vermutet, daß es eine solche Enzymaktivität nicht gibt. Mit EXSYMessungen konnte nicht nur diese Enzymaktivität nachgewiesen werden, sondern es gelang auch das verantwortliche Enzym, das Glutathion abhängiges FormaldehydaktivierendesEnzym (Gfa) genannt wurde, erstmals zu reinigen und das kodierende Gen zu identifizieren. (5) Die Anzahl der UbichinonBindungsstellen des Cytochrom bc 1 Komplexes aus Bos bovis wurde bestimmt. Dafür wurde eine NMRMethode entwickelt, die es ermöglicht, Bindungsstudien von wasserunlöslichen Cofaktoren an membranständigen Proteinen durchzuführen. Über die Aufnahme und Integration von 1 H, 13 CHSQCSpektren unter Verwendung von HRMASNMR konnte die Konzentration des Ubichinons, das in der Proteoliposomenmembran frei beweglich ist, quantifiziert werden. Verdrängungsstudien mit spezifischen Inhibitoren ermöglichten es dann, durch den Vergleich der freigesetzten Ubichinonkonzentrationen relativ zu der Cytochrom bc 1 Komplexkonzentration nachzuweisen, daß insgesamt drei Ubichinone spezifisch an den Cytochrom bc 1 Komplex binden. Die Ergebnisse werden in 5 Abschnitten beschrieben. Im Anhang zu jedem Abschnitt finden sich die Abdrucke der bereits erschienenen Veröffentlichungen (Abschnitte 1 bis 3) und ein zur Veröffentlichung akzeptiertes Manuskript (Abschnitt 5). In der Einleitung werden die Möglichkeiten aufgezeigt, mit modernen NMRspektroskopischen Methoden Protein LigandWechselwirkung zu studieren. In der Diskussion werden anhand der Ergebnisse die Limitierung, aber auch das noch nicht ausgeschöpfte Potential dieser Methoden erläutert. Während der Doktorarbeit wurden auch Untersuchungen zu den katalytischen Eigenschaften einer energiekonservierenden Hydrogenase aus M. barkeri durchgeführt. Die daraus entstandene Publikation ist im Anhang zu finden.
Hepatitis B caused by infection with the hepatitis B virus (HBV) still ranks among the most challenging infectious diseases of our time. Despite the availability of an effective prophylactic vaccine, 240 million people worldwide are estimated to be chronically infected with HBV and are at risk of developing life-threatening liver diseases, including cirrhosis and liver cancer. The underlying pathogenic mechanisms of HBV-associated liver diseases are only incompletely understood. It is widely accepted that liver pathology results from long-term immune-mediated liver injury and inflammation as a consequence of inefficient viral elimination. This injury can be naturally compensated by liver regeneration. However, chronic liver damage and permanent inflammation debilitates the regenerative capacity of the liver and fosters fibrosis as well as accumulation of chromosomal aberrations, which both contribute to cirrhosis and liver cancer. Liver regeneration requires the presence of the redox-sensitive transcription factor Nrf2 and intact insulin receptor signaling. A lack of Nrf2 causes increased intracellular levels of reactive oxygen species (ROS) that inactivate insulin receptor signaling and induce insulin resistance. Interestingly, HBV was observed to activate Nrf2 and the expression of Nrf2-regulated genes. This argues against an inhibitory effect of HBV on insulin receptor signaling by increased ROS levels. However, chronic HBV infection is associated with dysregulation of hepatocyte proliferation and retardation of liver regeneration. Hence, the aim of this thesis was to investigate the influence of HBV on the process of liver regeneration with respect to the insulin receptor signaling pathway. After short-term carbon tetrachloride (CCl4)-induced liver damage, HBV transgenic mice present prolonged liver damage and impaired liver regeneration as reflected by reduced hepatocyte proliferation and increased apoptosis. Impaired hepatocyte proliferation in HBV transgenic mice correlates with diminished activation of the insulin receptor. It was further observed in vitro that the activation of Nrf2 by HBV induces increased levels of the insulin receptor mRNA and protein in HBV-expressing cells. Strikingly, stably HBV-expressing cells as well as primary mouse hepatocytes from HBV transgenic mice bind less insulin due to reduced amounts of insulin receptor on the cell surface. This is caused by intracellular retention of the insulin receptor in HBV-expressing cells as a consequence of increased amounts of the cellular trafficking factor α-taxilin. The reduced amounts of insulin receptor on the cell surface impair insulin sensitivity in HBV-expressing cells and inactivate downstream signaling cascades that initiate insulin-dependent gene expression and glucose uptake. As a consequence of impaired hepatocyte proliferation and liver regeneration, HBV transgenic mice exhibit increased development of fibrosis after long-term CCl4-induced liver damage. Taken together, in this thesis, a novel pathomechanism could be uncovered that includes inactivation of insulin receptor signaling by HBV via intracellular retention of the insulin receptor leading to impaired liver regeneration after liver damage and promotion of liver fibrosis. These findings significantly contribute to an enhanced understanding of HBV-associated liver pathogenesis.