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Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
Cardiac progenitor cells hold great potential for regenerative therapies in heart disorders. However, the molecular mechanisms regulating cardiac progenitor cell expansion and differentiation remain poorly defined. Here we show that the multi- adaptor protein Ldb1, which mediates interactions between different classes of LIM domain transcription factors, is a multifunctional regulator of cardiac progenitor cell differentiation. Ldb1-deficient embryonic stem cells (ESCs) show a markedly decreased expression of second heart field (SHF) marker genes and subsequently impaired cardiomyocyte differentiation. Conditional ablation of Ldb1 in the early SHF using an Isl1-Cre driver led to embryonic lethality at Embryonic day (E)10.5 with cardiac abnormalities including a significantly smaller right ventricle and a shortened outflow tract, supporting a crucial role of Ldb1 in the SHF. Mechanistically we show that the importance of Ldb1 for SHF development is two-fold: On the one hand, Ldb1 binds to Isl1 and protects it from proteasomal degradation, as a consequence of which Ldb1-deficiency leads to an almost complete loss of Isl1+ cardiovascular progenitor cells. On the other hand the Isl1/Ldb1 complex promotes long-range promoter-enhancer interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing (3C- seq) identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes which play key roles in cardiac progenitor cell function and cardiovascular development. These interactions are of critical importance to regulate the expression of the downstream target genes since their expression levels are strongly dependent on the Ldb1/Isl1 levels. Overexpression of an Ldb1 mutant, which contains the LIM interaction domain and thereby can protect Isl1 protein from degradation, but lacks the dimerization domain and thus cannot promote long-range interactions, does not collaborate with Isl1 to regulate the expression of their common targets and results in defects in Isl1+ cardiac progenitor differentiation. In this thesis we show one of the first examples of genome-wide chromatin reorganization mediated by a developmental regulated, cell type specific, transcription complex. Ldb1 in concert with Isl1 promotes long range promoter- enhancer and enhancer-enhancer interactions in order to create active chromatin hub where gene important for heart development can be co-regulated. Moreover, Isl1 and Ldb1 genetically interact during heart development, as Isl1/Ldb1 haplodeficient embryos show various cardiac anomalies. The dosage-sensitive interdependence between Isl1 and Ldb1 in the expression of these key factors in cardiogenesis, further supports a key role of the Isl1/Ldb1 complex in coordinating a three dimensional genome organization, upstream of a regulatory network driving cardiac differentiation and heart development.
In conclusion, the Isl1/Ldb1 complex orchestrate a genome-wide three dimensional chromatin reorganization resulting in a transcriptional program responsible for the differentiation of multipotent cardiac progenitor cells into cardiomyocytes.
In der vorliegenden Arbeit galt es, stabile, lumineszente, tetrakoordinierte Organoborane unter Verwendung eines Bor-funktionalisierten ditopen Grundbausteins und unterschiedlicher π- konjugierter Ligandensysteme zu synthetisieren. Die Bifunktionalität sollte die gleichzeitige Einführung von zwei Lewis-Basen erlauben, um eine mögliche elektronische Kommunikation oder einen Energietransfer zwischen den Chromophoren zu gewährleisten.
...
Zusammenfassend war es möglich unter Einsatz eines Bor-haltigen Grundsystems (DBA) durch die Variation der chelatisierenden bzw. verbrückenden π-konjugierten Liganden stabile und effiziente Fluorophore mit nützlichen optischen Eigenschaften zu realisieren.
Small molecule drug discovery is strongly supported by biophysical data. In the reach of this thesis, cell free protein expression was used to produce human target proteins for ligand binding assays using Surface Plasmon Resonance spectroscopy (SPR). In the second step the binding and interaction characteristics of small molecules and fragments were analyzed using Nuclear Magnetic Resonance spectroscopy (NMR).
The first target protein was the human acid sensing channel 1 (ASIC1a). ASIC1a was expressed in a cell free expression system based on E.coli lysate. To optimize the expression, several parameters including fusion tags, ion concentrations and different hydrophobic environments were tested.
The adaption of the folding environment for ASIC1a needed more optimization, because it is a very challenging target to express in an in vitro system. Three different expression modes were employed to find a suitable folding environment.
SPR binding studies with ASIC1a were performed with chicken ASIC1a expressed in insect cells. The immobilization of cASIC1a and the used buffer conditions were tested using Psalmotoxin 1, a naturally occurring peptide venom which binds strong to the trimeric form of ASIC1a. Compound characterization experiments were performed with a variety of different ligands including amiloride, a general blocker of the whole ENaC protein family. None of the used ligands showed titration curves that would match a simple 1:1 binding model. The experiments either show no binding signal or signal that could be interpreted as unspecific binding. Even amiloride that should be binding the protein shows no signals that fit a simple binding model.
Another target protein that was investigated is the soluble prolyl cis/trans isomerase Cyclophilin D (or peptidyl prolyl isomerase F – PPIF). This protein is involved in the regulation of the mitochondrial permeability transition pore and therefore a potential drug target to treat neurodegenerative diseases. Small molecule binding was tested with CypD using SPR. Following the kinetic analysis of small molecule ligands, the binding position of different binding fragments was analyzed. These fragments originated from a SPR based fragment screen and gave no co-crystal structures with CypD. Therefore NMR was used to investigate the binding position of these fragments. An analysis of the chemical shift perturbations upon ligand addition revealed that the NMR analysis was in line with the results gathered by x-ray crystallography. The fragments with unknown binding position however, all bind to a specific patch slightly outside the binding pocket.
The ligand CL1 showed a special behavior in the NMR experiments. Upon addition to CypD, it produced large shifts on many signals of the protein, accompanied by a severe line broadening. The shift perturbations were so numerous and large that the spectrum had to be reassigned in complex with the ligand. Triple selective labeling was applied to allow a fast and nearly complete signal assignment. The possibility to use highly sophisticated labeling schemes, is one of the advantages of cell free protein expression. After the assignment of the complex spectrum, the chemical shift perturbations were analyzed and quantified. The residues showing the strongest CSPs are also identified in the crystal structure to be involved in the binding of CL1, giving a consistent picture. The numerous and large shift perturbations, produced by CL1 led to the assumption, that the ligand induces a conformational change in CypD, which is not represented in the co-crystal structure. This conformational change was characterized by a NMR based structure determination. CypD apo yielded a defined bundle, whose folded regions overlap well with the corresponding crystal structure.
For the calculation of the CypD-CL1 complex structure, the sidechain resonances were assigned using an automated assignment approach with the software FLYA. The calculation of the CypD-CL1 complex structure did not result in a defined bundle. While parts of the protein converge in a well folded state, the region around the active site shows no defined folding. Careful analysis of the structure calculation suggests that the problems during structure calculation did not originate from an incorrect resonance assignment, but rather from a lack of NOE crosspeaks. This might be due to a broadening of the corresponding NOE crosspeaks or the coexistence of many different conformations. This leads to the conclusion, that the protein conformation is not defined by the NMR data and could be in a dynamic interchange between multiple structures.
This hypothesis is supported by other observations. The line broadening of the signals in the complex is pronounced in the area around the active site and the substrate binding pocket, hinting to a connection between catalytic activity and protein dynamics. In addition many NMR signals are sensitive to changes in the measurement field strength and the temperature. This field dependent signal splitting suggests dynamic conformational changes in the protein between at least two different conformations on a millisecond timescale.
The current working model is that CL1 binds to CypD and induces the catalytic cycle and the connected conformational changes in CypD. As a result the proline like moiety in CL1 is constantly switching between the cis and the trans conformation. Due to the high affinity of CL1, the inhibitor does not leave the binding pocket after successful catalysis, but stays bound in the pocket stimulating further catalytic cycles. These findings as well as the working model are well in line with data published for Cyclophilin A, another member of the cyclophilin family, thereby supporting the model.
During the last decade of the 20th century, the field of mass spectrometry has seen a revolutionary change in its application and scope. The introduction of soft ionization methods for the analysis of biological molecules has expanded the area of mass spectrometry from its early roots in the analysis of inorganic and organic species into the fields of biology and medicine.
Today, the use of the mass spectrometry is extended to a wide range of applications in biotechnology and pharmaceutical industry, in geological, environmental and clinical research. In biochemistry, the principles of mass spectrometry are, however, broadly applicable in accurate molecular weight determination, reaction monitoring, amino acid sequencing, oligonucleotide sequencing and protein structure.
In order to carry out their biological activities, proteins interact most often to each other and form transient or stable complexes. In addition, some proteins specifically interact also with other proteins or with non-protein molecules, such as DNA, RNA or metabolites, these interactions being critical for their function. Hence, defining the composition of protein complexes, as well as understanding how protein complexes are assembled and regulated yield invaluable insights into protein function. Coupled with an isolation technique to purify a specific protein complex of interest, mass spectrometry can rapidly and reliably identify the components of complexes. In addition, quantitative MS techniques offer the possibility of studying dynamically regulated interactions....
Die Biosynthese der Fettsäuren (FS) ist in Eukaryoten und Bakterien ein hochkonserviert zentraler Stoffwechselweg, der in zwei strukturell verschiedenen Systemen ausgeführt wird. Die meisten Bakterien, Parasiten, Pflanzen und Mitochondrien nutzen ein Fettsäuresesynthase Typ-II (FAS-II) System. Bei FAS II Systemen sind alle katalytischen Domänen separate lösliche Proteine. In Eukaryoten wie auch den Bakterien Corynebakteria, Mycobakteria, Nocardia (Klasse der CMN Bakterien) liegen die katalytischen Domänen fusioniert auf einer Polypeptidkette vor, die zu einem Multienzymkomplex der Fettsäuresynthase Typ I (FAS-I) assemblieren. Die Architektur der FAS-I zeigt große Unterschiede; die X förmige Säuger-FAS-I (Maier et al., 2006), sowie die fassartigen Enzyme der Pilz FAS-I (Jenni et al., 2007; Leibundgut et al., 2007; Lomakin et al., 2007; Johansson et al., 2008) und der bakteriellen FAS-I (Boehringer et al., 2013; Ciccarelli et al., 2013). Zwischen Pilz- und bakterieller FAS-I gibt es trotz des ähnlichen Aufbaus bedeutende Unterschiede. Mycobakterium tuberculosis, der Auslöser von Tuberkulose (TB), an der jährlich über eine Million Menschen weltweit sterben (WHO, 2014), synthetisiert durch eine Symbiose von FAS-I, FAS-II und der Polyketidsynthase-13 Mykolsäuren. Durch die Mykolsäuren ist M. tuberculosis resistent gegen äußere Einflüsse. FAS-I ist in die Synthese der Vorstufen der Mykolsäuren involviert. Sie stellt im Kampf gegen TB ein potentielles Inhibierungstarget dar.
Strukturell war die bakterielle FAS-I beim Beginn der vorliegenden Arbeit, nur durch negative-stain-Elektronenmikroskopie (EM) Aufnahmen aus dem Jahr 1982 charakterisiert (Morishima et al., 1982). In dieser Arbeit konnte die bakteriellen FAS I aus M. tuberculosis (MtFAS), sowie Corynebacterium ammoniagenes (CaFAS) und Corynebacterium efficiens (CeFAS) strukturell untersucht werden. Dies geschah mit den Methoden negative-stain-EM, Einzelmolekül-Cryo-EM (Cryo-EM), Cryo EM Tomographie (CET) und Röntgenkristallographie.
Anhand von CeFAS-Kristallen konnte erstmals durch Röntgenkristallographie die Struktur einer bakteriellen FAS-I bestimmt werden. Zudem wurde die hohe konformationelle Flexibilität der bakteriellen FAS-I mit mehreren Methoden gezeigt. Für die CaFAS konnte mit Cryo-EM initiale Prozesse der Proteinkristallbildung abgebildet werden.
A novel series of ribonucleosides of 1,2,3-triazolylbenzyl-aminophosphonates was synthesized through the Kabachnik–Fields reaction using I2 as catalyst followed by copper-catalyzed cycloaddition of the azide–alkyne reaction (CuAAC). All structures of the newly prepared compounds were characterized by 1H NMR, 13C NMR, and HRMS spectra. The structures of 2e, 2f, 3d, and 3g were further confirmed by X-ray diffraction analysis. These compounds were tested against various strains of DNA and RNA viruses; compounds 4b and 4c showed a modest inhibitory activity against respiratory syncytial virus (RSV) and compound 4h displayed modest inhibitory activity against Coxsackie virus B4.
Rotary adenosine triphosphate (ATP)ases are ubiquitous, membrane-bound enzyme complexes involved in biological energy conversion. The first subtype, the so-called F1Fo ATP synthase, predominantly functions as an ATP synthesizing machinery in most bacteria, mitochondria and chloroplasts. The vacuolar subtype of enzyme, the V1Vo ATPase, operates as an ATP driven ion pump in eukaryotic membranes. The subtype found in archaea and some bacteria is called A1Ao ATP (synth)ase and is capable of working in both directions either to synthesize ATP or to generate an ion motive force by consuming the same.
All the three above-mentioned subtypes of rotary ATPases work as nanomolecular machines sharing a conserved mechanism to perform the energy conservation process. The simplest form of these enzymes is the bacterial F1Fo ATP synthase. Here, ions are channelled via the membrane stator subunit a to the rotor ring of the enzyme. After almost a complete rotation of the ring the ions are released again on the other side of the membrane. This rotation is further transmitted via the central stalk to the soluble part of the enzyme, the F1-complex, where conformational changes within the nucleotide binding sites result in the synthesis of ATP from ADP and Pi.
The rotor or c-ring of the enzyme is the key protein complex in mediating transmembrane ion translocation. Several structural and biochemical methods have been applied in the past years to study the rotor rings from many different organisms. The results revealed that the stoichiometry of a c-ring of a given species is constant while it can vary between different species within a range of 8 to 15 c subunits. The c-ring stoichiometry determines directly the number of ions transported through Fo per rotation whereby three molecules of ATP are concurrently synthesized in the water-soluble F1 headgroup. Hence the number of c subunits has an important influence on the bioenergetics of the corresponding enzyme and thus the entire organism.
The c-ring of a rotary ATPase is able to specifically bind either protons (H+) or sodium ions (Na+) as the coupling ion for the enzyme. Several structures are already available revealing the coordination network of both types of rotor rings. In each case ion binding includes a highly-conserved carboxylic acid residue (glutamate or aspartate), in addition to a more varying combination of amino acid residues, whereby Na+ coordination is structurally more demanding than H+ binding.
In the first part of my PhD thesis, I aimed to characterize the F1Fo ATP synthase rotor ring of the opportunistic pathogenic bacterium Fusobacterium nucleatum on a functional and structural level. F. nucleatum is an anaerobic bacterium which uses peptides and amino acids as a primary energy source. It is one of the most frequently occuring bacteria in human body infections and involved in human periodontal diseases.
The protein complex was heterologously expressed within a hybrid ATP synthase in Escherichia coli and purified without an affinity tag for further analysis. Two high resolution X-ray structures of the c-ring were solved at low (5.3) and high (8.7) pH to 2.2 and 2.64 Å, respectively. In both structures, the conserved glutamate is in an ion-locked conformation, revealing that the conformational state of the ion binding carboxylate is not depending on the pH of the crystallization condition, which is in good agreement with previous structural and biochemical studies of other c-rings.
A Na+ ion is present within the c-ring binding site and directly coordinated by four amino acid residues and a structural water molecule. Remarkably, the Na+ is bound by two glutamate residues instead of one as is the case in the I. tartaricus Na+ binding c-ring, of which the first high resolution X-ray structure of a c-ring has been solved in 2005. Thus, a new type of Na+ coordination in an ATP synthase rotor ring with a two-carboxylate ion binding motif is described here, which also occurs in other bacteria, including several pathogens. Na+ specificity of the investigated c-ring was further confirmed by a competitive biochemical labeling reaction performed with a fluorescent ATP synthase inhibitor molecule (N-cyclohexyl-N`-[4(dimethylamino)-α-naphtyl] carbodiimide, NCD-4).
We furthermore complemented our functional and structural data of the F. nucleatum c-ring by computational studies to explore the ion translocation mechanism of this enzyme in more details. We therefore analyzed the protonation state of the second, additional glutamate in the ion binding site. Molecular dynamics (MD) simulations and free-energy calculations indicated that this glutamate is constitutively protonated, in the ion-locked as well as in a simulated, more hydrated open-conformation of the ion binding glutamate as when it is travelling through the a/c-ring interface upon c-ring rotation.
During my thesis, I worked on two different membrane proteins. One is a bacterial secondary transporter and the second is a human mitochondrial calcium channel.
The first part of my thesis involves the structural and biochemical characterization of an L-carnitine/ γ-butyrobetaine antiporter from bacteria called CaiT. The aim of the project was to understand the Na+ independence of CaiT and to determine the crystal structures of CaiT in different conformations to expand the mechanistic understanding of substrate/ product antiport in CaiT.
The study revealed how a positively charged amino acid side chain (arginine 262) in CaiT could structurally and functionally mimic a sodium ion. Additionally, various crystal structures of CaiT obtained in this study demonstrate that the central substrate-binding site is highly dynamic and can accommodate the substrate in various orientations.
In the second part of my thesis, I was able to optimize the expression and purification conditions for the human mitochondrial calcium uniporter or the MCU. Understanding how this channel functions can help us unravel the mechanism of calcium uptake by mitochondria. Secondary structure prediction analysis in combination with mass spectrometry of degraded MCU products obtained during the purification of the full-length protein led to the identification of a stable MCU construct. This study resulted in the successful purification of milligram quantities of stable MCU protein for the first time. Further optimization may be required to obtain more homogenous protein that is amenable for crystallization.
Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.