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Nukleinsäuren und Proteine bilden zusammen mit den Kohlenhydraten und Lipiden die vier großen Gruppen der Biomoleküle. Dabei setzen sich Nukleinsäuren aus einer variierenden Abfolge von Nukleotiden zusammen. Gleiches trifft auf die Proteine zu, wobei deren Bausteine als Aminosäuren bezeichnet werden. Die Reihenfolge der Bausteine bestimmt zusammen mit der Interaktion, die die einzelnen Bestandteile untereinander eingehen, deren Funktion. Um deren Wirkungsweise verstehen und nachverfolgen zu können, wurden unterschiedliche Methoden entwickelt, zu welchen auch die EPR-Spektroskopie gehört.
Durch den Einbau modifizierter Nukleotide oder Aminosäuren lassen sich Spinlabel in die sonst EPR-inaktiven Nukleinsäuren und Proteine einführen. Diese Marker lassen sich grundsätzlich in drei Klassen unterteilen (Metallionen, Nitroxidradikale und TAMs), weisen aber immer mindestens ein ungepaartes Elektronenpaar auf. Die Festphasensynthese ist eine Standardprozedur zur Herstellung von markierten Nukleinsäuren und Proteinen. Allerdings führen die Bedingungen dieser Methode zumindest teilweise zur Zersetzung der Nitroxidradikale, die dieser Arbeit zugrunde liegen, wenn sie direkt während der Synthese eingebaut werden. Der direkte Einbau ist aber in vielen Fällen essenziell, um bestimmte Eigenschaften zu erzielen.
Um den Abbau des Nitroxidradikals während der Festphasensynthese zu verhindern, kann dieses vorübergehend mit einer Schutzgruppe versehen werden, welche sich anschließend wieder abspalten lässt.
Der Schwerpunkt dieser Arbeit liegt hierbei auf der Darstellung neuer photolabil geschützter Spinlabel zur Synthese markierter Proteine und Nukleinsäuren.
Basierend auf den Nukleotiden Uridin und Cytidin konnten zwei für die RNA-Synthese vorgesehene Phosphoramidite synthetisiert werden, welche jeweils an der 5-Position des Pyrimidinrings mit einem photolabil geschützten Spinlabel auf Basis von TPA versehen waren. Durch Einbau des Uridinderivats in das Neomycin-Aptamer konnte zudem der Einfluss der Spinlabel auf die lokale Struktur mit Hilfe von in-line probing gezeigt werden.
Der gleiche TPA-Label konnte ebenfalls mit einem Lysin gekuppelt werden, welches später über ein orthogonales tRNA/Aminoacyl-tRNA Synthetase Paares in eine Polypeptid eingebaut werden sollte. In Kooperation mit dem AK Grininger ist auch ein nicht geschützter Spinlabel zur kupferfreien Markierung der Fettsäuresynthase entstanden. Abschließend war noch die Synthese eines auf Phenylalanin basierenden photolabil geschützten Spinlabel in Arbeit, welcher jedoch nicht beendet werden konnte. Dieser sollte mittels Festphasensynthese einbaubar sein, weswegen er am N-Terminus mit Fmoc geschützt ist.
Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared to the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans hermaphrodite cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on acetylcholine and neuropeptides (Steuer Costa et al., 2017), are altered like in animals with reduced cAMP. Synapsin mutants have slight alterations in synaptic vesicle (SV) distribution, however, a defect in SV mobilization was apparent after channelrhodopsin-based photostimulation. DCVs were largely affected in snn-1 mutants: DCVs were ∼30% reduced in synaptic terminals, and not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. SNN-1 co-localized with immobile, captured DCVs. In synapsin deletion mutants, DCVs were more mobile and less likely to be caught at release sites, and in non-phosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced SNN-1 dependent tethering. Our work establishes synapsin as a key mediator of neuropeptide release.
Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.
Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.
Specialized transporter proteins facilitate controlled uptake and extrusion of molecules across biological membranes that would otherwise be impermeable to them. The superfamily of solute carriers (SLC) comprises the second largest group of membrane proteins in humans, acting on a variety of small polar and non-polar molecules and ions. Because of their central role in metabolism, malfunctioning of these proteins often is pathogenic. The interest in SLC transporters as drug targets – as well as for drug delivery – has therefore increased in the past years. For many SLC subfamilies, however, structural and functional information remains scarce to date.
The here presented data provides important insights into different aspects of the transport mechanism of the SLC23 and SLC26 protein families. Importantly, we show that SLC23 nucleobase transporters, in contrast to what was been previously reported, work as uniporters rather than as proton-coupled symporters. In order to do so, we developed the first and only in vitro transport assay for the SLC23 family, which enables investigation of protein function in a defined environment. Moreover, we provide a hypothesis on the role of the extremely conserved negative charged substrate binding site residue found not only in the SLC23, but also SLC4 and SLC26 families. Based on a detailed analysis of binding and transport we conclude that this conserved negative charged has a relevance for protein stability rather than for substrate binding, which explains its conservation for all three protein families that otherwise differ in their substrate specificities and modes of transport. Lastly, we investigated the relevance of oligomerization for the SLC23 and SLC26 families, highlighting the importance of the STAS domain for forming active dimers in the SLC26 anion transporter family.
The enzyme 5-lipoxygenase (5-LO) occupies a central role in the biosynthesis of inflammatory leukotrienes and thus takes part in the pathogenesis of related diseases. Its occurrence is mainly restricted to cells of the immune system including granulocytes, monocytes/macrophages or B-lymphocytes and can be induced by cell differentiation of myeloid cells after treatment with differentiating agents, such as DMSO, retinoic acid or the combination of TGFβ/1,25(OH)2D3. The latter contribute to the highest level of induction of mRNA and protein expression. Its cell specific occurrence is at least partly due to DNA methylation in cells that do not exhibit 5-LO activity and genetic regulation is further dependent on histone acetylation. 5-LO expression is controlled by transcription factors binding to the promoter sequence of the ALOX5 gene that induce basal promoter activity, as well as promoter independent effects including transcript initiation and elongation, which are mostly attributed to TGFβ/1,25(OH)2D3 signaling. The ALOX5 gene resembles a typical housekeeping gene, hence lacks TATA- or CAAT-boxes for transcriptional regulation, but displays a high GC-content with eight GC-boxes, five of which are arranged in tandem, that provide binding sites for transcription factors Sp1, Sp3 and Egr-1.
The proximal ALOX5 promoter is furthermore a target for additional factors, such as TGFβ effector proteins SMADs or the vitamin D receptor and possesses additional consensus sequences for transcriptional regulators, including NF-κB or PU.1. However, as yet no actual binding of these proteins to the promoter sequence was demonstrated and an unbiased screening for identifying further ALOX5 promoter interacting proteins, which might have impact on 5-LO expression, is still lacking. For this purpose, the present study focused on the identification of significantly interacting proteins, employing DNA-affinity enrichment coupled to label-free quantitative proteomics, spanning a sequence of about 270 base pairs of the proximal ALOX5 promoter. For the elucidation of potential cell specific differences in protein patterns and compositions, DNA pulldowns were performed by using oligonucleotide stretches comprising the core promoter sequence including the 5-fold GC-box, which were incubated with different cell lines and differentiation states of myeloid, as well as B-lymphocytic lineages. In order to compare different mass spectrometric quantification strategies that would allow for identification of interactors, dimethyl labeling and label-free techniques were used. Since the label-free approach outperformed the label-based one in initial experiments, it was established as standard quantification strategy in all DNA pulldowns performed. The pulldowns of myeloid cell lines in both undifferentiated and differentiated state and B-lymphocytes resulted in a cell-unspecific protein pattern whose composition was similar, regardless of cell lineage. Additionally, further DNA sequences comprising either a vitamin D response element or a SMAD binding element were investigated in the promyelocytic model cell line HL-60 in both undifferentiated and differentiated state. The identified proteins confirmed known interaction partners and furthermore revealed novel potential regulators of the 5-LO promoter. Out of these, the most prominently identified and promising proteins included transcription factors of the KLF- and CCAAT/enhancer binding protein-family. In this context, KLF5 and KLF13 are both involved in the regulation of inflammatory processes, the former additionally being an effector protein of TGFβ-signaling, whose functional characterization is of utmost interest in terms of regulation of 5-LO expression. Further protein characterization will be inevitable for the CCAAT/enhancer binding proteins C/EBPα, C/EBPβ and C/EBPε. These transcription factors are involved in the regulation of inflammatory processes and heterodimers thereof (C/EBPα/β) are known to control TGFβ/1,25(OH)2D3-mediated effects of the CD14 gene.
Several of the identified proteins of the pulldowns containing the tandem GC-box represented interactors of G-quadruplex DNA, including the helicases BLM and DHX36, the ribonucleoproteins hnRNP D and hnRNP K and transcription factor MAZ. Since G-quadruplexes form in G-rich DNA sequences as secondary DNA structures and exhibit substantial regulatory effects on the transcription of their target genes, the potential formation thereof in the ALOX5 core promoter sequence was investigated in a second project. Out of the proteins mentioned above, MAZ is shown to exert resolving effects on G4-DNA and synergistically induce Sp1-dependent gene activation of oncogene h-RAS, which displays analogous promoter characteristics to the ALOX5 gene. A DNA stretch comprising the tandem GC-box was used for elucidating the potential of secondary DNA structure formation. Intriguingly, both immune-based and spectroscopic methods provided clear evidence for the in vitro G-quadruplex formation of the proximal promoter sequence for the first time. In order to provide additional information on a possible regulatory effect of existing G-quadruplex structures on 5-LO transcription, differentiated HL-60 cells were subsequently treated with two distinct G4-DNA stabilizing agents. A porphyrin analogon (TMPyP4) did not exhibit any effects on 5-LO mRNA and protein expression after cell treatment. A second G4-DNA stabilizing agent (pyridostatin) on the other hand revealed significant reduction on 5-LO protein expression after cellular treatment. These mixed results render further experiments inevitable, in order to provide a clear assertion as to whether 5-LO expression is regulated by G-quadruplex structures or not.
Altogether, this study enlarges the knowledge of ALOX5 proximal promoter interacting proteins by corroborating the binding of already known transcription factors and identifying novel interactors. It yields essential groundwork for subsequent functional studies of proteins involved in 5-LO transcription and introduces G-quadruplexes as a new potential mechanism in ALOX5 gene regulation.
Structure-function relationships in substrate binding protein dependent secondary transporters
(2023)
This work provides new insights into the relevance of SBP dependent secondary transport systems, especially in the thus far under-researched subgroup of TAXI transporters. Importantly, we identified and characterized the TAXI transport system TAXIPm-PQM from Proteus mirabilis. We demonstrated that, in contrast to previously characterized SBP dependent secondary transport systems, TAXIPm-PQM is a proton coupled system and transports the C5-dicarboxylate α- ketoglutarate. Since initially the transport of α-ketoglutarate could only be demonstrated in vivo but not in vitro using established protocols (Mulligan et al. 2009), we investigated in detail the differences between the in vivo and in vitro assay. This resulted in a bioinformatic analysis of TRAP and TAXI signal peptides, which strongly implied that TAXIPm-P requires a transmembrane anchor to allow for transport. We then provided TAXIPm-P surface tethered to the membrane in in vitro transport assays and confirmed the prediction of our bioinformatic analysis that TAXIPm-PQM deploys a membrane-anchored instead of a soluble SBP. Furthermore, the TAXI transport system TAXIMh-PQM from Marinobacter hydrocarbonoclasticus transports fumarate only if both membrane domains Q and M are present. For further characterization, Michaelis-Menten kinetics and affinities were determined for both TAXI transport systems TAXIPm-PQM from Proteus mirabilis and TAXIMh-PQM from Marinobacter hydrocarbonoclasticus. In addition, nanobodies were selected for the membrane domain TAXIPm-QM from Proteus mirabilis to stabilize different conformations which can serve in subsequent structural elucidation studies. Furthermore, the TRAP SBP TRAPHi-SiaP from Haemophilus influenzae was shown to interact not only with its corresponding membrane domain TRAPHi-SiaQM but with at least one additional transporter. It was thereby excluded that TRAPHi- SiaP transfers N-acetylneuraminic acid to the only native E. coli TRAP transporter TRAPEc-YiaMNO and suggested to rather interact with a SBP dependent ABC transport system as this protein family represents the largest SBP dependent protein group in E. coli (Moussatova et al. 2008).
The transcriptional regulator RcsB controls the expression of a minimum of 20 different genes having diverse functionalities and biosynthetic operons in the family of Enterobacteriaceae. While in the heterodimeric complex with the co activator RcsA, the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various co activators, resulting in recognition of different DNA targets. In this study the interaction of RcsB with some of these DNA targets have been analysed by a diverse array of techniques including gel shift assay and SPR. The solution structure of the C-terminal DNA-binding domain of RcsB from Erwinia amylovora spanning amino acid residues 129-215 has been solved in this study by heteronuclear NMR spectroscopy. The C-terminal domain is composed of four α-helices where the two central helices of the H-T-H motif are similar to the structures of the regulatory proteins GerE, NarL and TraR. The DNA-binding activity of the C-terminal domain alone is established for the first time in this study and was specified by fluorescence spectroscopy, SPR and NMR titration experiments. The molecular interaction between the individual RcsB domains was analysed by cross-linking experiments and heteronuclear NMR spectroscopy and the amino acid residues of the C-terminal domain involved in this interaction were identified precisely. Another important part of this project was the cell-free production of different Trp analogue labelled RcsB protein. RcsB protein was produced in quite a good yield with different Trp analogue having spectrally enhanced properties. The isolated RcsB alloproteins proved to be ideal for protein interaction studies by fluorescence spectroscopy and the very first evidence of an oligomerization of RcsB due to molecular association has been put forth from these studies. The phosphorylated state of the RcsB protein was mimicked by a beryllofluoride complex in order to study its role in transcriptional regulation. It was found that RcsB alone could bind to DNA targets upon this modification by the beryllofluoride complex. Thus the phosphorylation of the protein that involves the Asp 56 residue induces a structural change of the protein followed probably by a domain movement also, so that the C-terminal domain having the H-T-H DNA binding motif that was previously eclipsed by the N-terminal domain is relieved of this constraint.
Salt-inducible kinases (SIKs) are key metabolic regulators. Imbalance of SIK function is associated with the development of diverse cancers, including breast, gastric and ovarian cancer. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based PAK inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.
H/ACA-RNPs are involved in RNA guided pseudouridylation of rRNAs and snRNAs. In this thesis I reconstituted active and labeled archaeal as well as eukaryotic H/ACA-RNPs and studied the structural dynamics of complex assembly and pseudouridine formation. Single molecule FRET spectroscopy was used as method of analysis to study structure, assembly and dynamics of these important complexes.