Refine
Year of publication
- 2017 (58) (remove)
Document Type
- Article (45)
- Doctoral Thesis (13)
Language
- English (58) (remove)
Has Fulltext
- yes (58)
Is part of the Bibliography
- no (58)
Keywords
- crystal structure (6)
- Research article (4)
- Biochemistry (3)
- Cell biology (3)
- Biophysics and structural biology (2)
- NMR spectroscopy (2)
- Optogenetics (2)
- RNA (2)
- Schiff bases (2)
- benzoxazines (2)
Institute
- Biochemie und Chemie (58) (remove)
A great challenge in life sciences remains the site-specific modification of proteins with minimal perturbation for in vitro as well as in vivo studies. Therefore, different chemoselective reactions and semi-synthetic techniques such as native chemical ligation or intein-mediated protein splicing have been established. They enable a site-specific incorporation of chemical reporters into proteins, such as organic fluorophores or unnatural amino acids. In this PhD Thesis, protein trans-splicing was guided by minimal high-affinity interaction pairs to trace proteins in mammalian cells. In addition, the temporal modulation of cellular processes by photo-cleavable viral immune evasins was achieved.
Protein trans-splicing mediated by split inteins is a powerful technique for site-specific and 'traceless' protein modifications. Despite recent developments there is still an urgent need for ultra-small high-affinity intein tags for in vitro and in vivo approaches. So far, only a very few in-cell applications of protein trans-splicing are reported, all limited to C-terminal protein modifications. Here, a strategy for covalent N-terminal intein-mediated protein labeling at sub-nanomolar probe concentrations was developed. Combined with the minimalistic Ni-trisNTA/His-tag interaction pair, the affinity between the intein fragments was increased 50-fold (KD ~ 10 nM). Site-specific and efficient 'traceless' protein modification by high-affinity trans-splicing is demonstrated at nanomolar concentrations in mammalian cells.
High background originating from non-reacted, 'always-on' fluorescent probes still is a crucial issue in life sciences. Covalent labeling approaches with simultaneous activation of fluorescence are advantageous to increase sensitivity and to reduce background signal. Therefore, high-affinity protein trans-splicing was combined with fluorophore/quencher pairs for online detection of covalent N-terminal protein labeling in cellular environments. Substantial fluorescence enhancement at nanomolar probe concentrations was achieved. This ultra-small fluorogenic high-affinity split intein system is an unprecedented example for real-time monitoring of the trans-splicing reaction in cell-like environments as well as for protein labeling with fluorogenic probes at nanomolar concentrations.
To extend the field of chemical immunology and to address spatiotemporal aspects in adaptive immune response, new tools to control antigen processing are required. Therefore, synthetic photo-conditional viral immune evasins were designed to modulate antigen processing on demand. By using light, the time and dose controlled antigen translocation by the transporter associated with antigen processing (TAP) was triggered with response in the second regime. Peptide delivery and loading by the peptide-loading complex (PLC) was rendered inactive, whereas blocking was abolished in a light-controlled fashion to inactivate the synthetic viral immune evasin ICP47 along with simultaneous activation of the antigen presentation pathway. Lightresponsive peptide translocation by the TAP complex was assayed in vitro by utilizing microsomes isolated from professional antigen presenting B-cell lymphomas (Raji). To extend these studies, suppression and photo-controlled rescue of antigen presentation was examined at single-cell resolution in human primary immune cells.
Native chemical ligation interconnects peptide chemistry with recombinantly expressed proteins. This technique was applied to generate the semi-synthetic full-length ICP47. Although this approach was realized, the low product yield was not sufficient for further functional studies. Therefore, full-length ICP47 was consecutively generated by utilizing a full synthetic four-fragment ligation approach. However, this synthetic viral immune evasin was not able to block peptide translocation in a robust way.
The centerpiece of all neuronal processes is the synaptic transmission. It consists of a complex series of events. Two key elements are the binding of synaptic vesicles (SV) to the presynaptic membrane and the subsequent fusion of the two membranes. SV are neurotransmitter-filled membranous spheres with many integral and peripheral proteins. The synaptic SNARE complex consists of three interacting proteins, which energize and regulate the fusion of the SV membrane with the presynaptic membrane. Both processes are closely orchestrated to ensure a specific release of neurotransmitter. Already many experiments have been performed, such as genetic screens and proteome analysis of SV, to determine the functions of the various proteins involved. Nevertheless, the functions of the identified proteins are still not fully elucidated. The aim of this thesis was initially applying a tandem affinity purification (TAP) of SV to identify unknown interaction partner of SV and to determine their role. This was supposed to be performed in the model organism Caenorhabditis elegans (C. elegans). The underlying mechanisms are conserved throughout the phylogentic tree and identified interaction partners will help to understand the processes in the mammalian brain. Although there is no neuron-rich tissue in C. elegans as in other model organisms, the diverse genetic methods allows a rapid creation of modified organisms and a prompt determination of the function of identified proteins. The integral SV protein synaptogyrin has been fused to a TAP-tag. The TAP-tag consists of a ProteinA, a TEV protease cleavage site and a calmodulin binding peptide (CBP). Both affinity purification steps are performed sequentially and allow a highly specific native purification of proteins and their interaction partners. Due to technical difficulties the purification strategy was modified several times during the course of this thesis and then finally abandoned for a more promising project, the SNARE complex purification. In conclusion, one of the reasons was the necessary lack of detergent.
The amended aim of this thesis has been the TAP of solubilized SNARE complex to identify unknown interaction partner and to determine their role. In order to increase the specificity of the purification, in terms of formed complexes, the two SNARE subunits, synaptobrevin (SNB-1 in C. elegans) and syntaxin (UNC-64 in C. elegans), were separately fused to the different affinity tags. As the modifications of the proteins could impair their function and lead to false interaction partners, their functionality was tested. For this purpose, the corresponding fusion constructs were expressed in strains with mutated snb¬1 and unc-64. Non-functional synaptic proteins display an altered course of paralysis in an aldicarb assay. The fusion proteins which were expressed in their respective mutant strains displayed a near to wild-type (WT) behavior in contrast to the naive mutant strains. Multiple TAP demonstrated SNB-1 signals in Western blot analysis and complex sets of proteins in the final elution step in a silver staining of SDS-PAGEs. These samples were sent with negative control (WT purification) for MS analysis to various cooperation partners. 119 proteins were identified which appeared only in data sets with SNARE proteins and not in WT samples. If proteins were detected in ≥ 2 SNARE positive MS analysis and had known neural functions or homologies to neuronal proteins in other species, they were selected for further analysis. These candidates were knocked down by RNAi and tested for synaptic function in a following aldicarb assay. The treatment with their specific RNAi resulted for mca-3 in a strong resistance, while frm-2, snap-29, ekl-6, klb-8, mdh-2, pfk-2, piki-1 and vamp-8 resulted in hypersensitivity. The most responsive genes frm-2, snap-29 and mca-3 were examined, whether they displayed a co-localization together with synaptobrevin in promoter fusion constructs or functional fusion constructs. In fluorescence microscopy images only MCA-3::YFP demonstrated neuronal expression.
In order to substantiate the synaptic nature and functionality of the MCA-3::YFP a swimming assay was performed. Here, fusion construct expressing strains, which contained mutated mca-3, were compared with untreated mutant strains and WT strains according to their behavior. In this swimming assay a partial restoration of WT behavior was shown in the MCA-3::YFP expressing mutant strains. Based on these data, we discovered with MCA 3 a new interaction partner of the SNARE complex. MCA-3 is a plasma membrane Ca2+-ATPase and was initially seen only in their role in the endocytosis. Its new putative role is the reduction of Ca2+ concentration at the bound SNARE complex. Since an interaction of syntaxin with Ca2+ channels has been demonstrated, it would be comprehensible to reduce the local concentration of Ca2+ to a minimum by tethering Ca2+ transporters to the SNARE complex.
Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.
This thesis is concerned with quantum dynamical propagation methods suitable for high-dimensional systems, and their application to excitation energy transfer (EET), electron transfer (ET), and intra-molecular vibrational redistribution (IVR) in molecular aggregates. The theoretical description of these processes, which are often ultrafast – with time scales in the range of femtoseconds to picoseconds – is challenging, both with regard to quantum dynamical simulations and electronic structure calculations.
The present thesis comprises two parts. The first part concerns the implementation of a novel quantum dynamical method based on Gaussian Wavepackets (GWPs): the 2-Layer Gaussian-MCTDH (2L-GMCTDH) method. This method, which has recently been proposed in [S. Römer, M. Ruckenbauer, I. Burghardt, The Journal of Chemical Physics, 2013, 138, 064106] was implemented in a Fortran90 code and applied to various high-dimensional test systems. The second part of the thesis addresses the combined electronic structure and dynamical study of a novel type of donor-acceptor systems that have been investigated in a joint project with experimental collaboration partners at Strasbourg University. In both parts, numerical applications focus on high-dimensional model Hamiltonians for EET and ET processes.
Regarding the first part, the interest of using GWP-based methods is two-fold: First, GWPs represent spatially localized basis sets that are useful for on-the-fly dynamics in conjunction with electronic structure calculations. Second, they are naturally suited for the explicit representation of quantum mechanical system-bath type problems where a large number of vibrational bath modes are weakly perturbed from equilibrium. In this context, various methods exist that are based upon classically evolving GWP bases. A major improvement results from variational methods which involve optimized, non-classical GWP trajectories. In particular, the variational Gaussian-based Multi-Configuration Time-Dependent Hartree (GMCTDH) and its variational Multi-Configurational Gaussians (vMCG) variant were originally derived as semiclassical variants of the Multi-Configuration Time-Dependent Hartree (MCTDH) method. However, the G-MCTDH and vMCG methods mostly use Frozen Gaussian (FG) basis sets that are far less flexible than the single-particle (SPF) representation of standard MCTDH. As a consequence, a significantly larger number of GWPs are generally required to reach convergence. To remedy the lack of flexibility of the FG basis sets, the abovementioned two-layer (2L-G-MCTDH) approach has been introduced: Here, the first layer is composed of flexible SPFs, while the second layer is composed of low-dimensional FGs. The numerical scaling properties are significantly improved as compared with the conventional G-MCTDH and vMCG schemes. The first implementation of the method in an in-house Fortran90 code is presented, along with applications to (i) a model of site-to-site vibrational energy flow in the presence of intra-site vibrational energy redistribution (IVR) and (ii) a multidimensional donor-acceptor electron transfer system described within a linear vibronic coupling model. The second system relates to a model for ET at an oligothiophene-fullerene interface relevant to organic photovoltaics. Besides the description of the implementation, a detailed assessment of the convergence properties and comparison with multi-layer MCTDH (ML-MCTDH) benchmark calculations is presented. Finally, a perspective is given on the future combination with the existing ML-MCTDH scheme; indeed, such a combination is straightforward since the first layer of the 2L-G-MCTDH approach can be chosen to be orthogonal.
Regarding the second part of the thesis, two generations of a novel donor-acceptor (DA) system for organic photovoltaics applications, involving self-assembled block co-oligomers DA dyads and triads with perylene-diimide (PDI) accepter units, are addressed within a collaborative project with S. Haacke and S. Mery (University of Strasbourg). Based upon detailed excited-state electronic structure investigations along with quantum dynamical and kinetic studies, the relevant ET formation and recombination steps are characterized quantitatively, in view of optimizing the chemical design and reducing recombination losses.
In a first-generation variant of the abovementioned DA systems, which involves liquid-crystalline triads, we were able to show that a highly efficient inter-chain ET process prevails over intra-molecular ET, leading to fast recombination. Due to the latter, this system turns out to be inefficient for photovoltaic applications. To fully understand the elementary steps, high-dimensional quantum dynamics simulations were carried out using the ML-MCTDH method, in collaboration with Matthias Polkehn from our group. In the second-generation variant, which is in the focus of the present thesis, both the nanomorphology and the chemical design were modified. The present work, focuses upon the aspect of chemical design, by characterizing a series of modified DA’s, with donor units of varying length while the PDI accepter units remain unchanged. The intra-molecular ET is observed in these systems, but the processes are comparatively slow, of the order of tens to hundreds of picoseconds. Hence, a kinetic analysis using the Marcus-Levich-Jortner rate theory is employed. Among the main results of the study is that addition of an electron donating amine unit strongly increases the lifetime of the charge-separated state, and therefore reduced recombination losses.
Overall, the present thesis shows how a combination of high-dimensional quantum dynamics, electronic structure calculations, and vibronic coupling model Hamiltonians can be employed to obtain an accurate picture of EET, ET, and IVR in high-dimensional molecular assemblies. Furthermore, the 2L-GMCTDH method paves the way for accurate and efficient on-the-fly calculations; a suitable set-up for such calculations is currently in progress.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the "assembly-line synthetic concept" of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.
If the biotechnological production of chemicals can further replace or support regular synthetic chemistry, industry will be able to move away from fossil oils towards renewable sources. However, in many cases the much needed adaptation of biotechnological production systems is not yet developed to the necessary level.
For processes where short fatty acids (FA) are needed, as for example in the microbial production of biofuels in the gasoline range, protein engineering had not yet delivered feasible solutions. In this thesis, several approaches to introduce chain length control on type I fatty acid synthases (FAS) were established and made available in a publication and two patents. Therein, engineering was focused on rational design based on available structural information.
First, the type I FAS from C. ammoniagenes was used as a model enzyme to probe modifications on FAS in a low complex in vitro environment in order to gain information about structure-function relationships. At this stage, engineering was conducted in several rounds, first addressing possible ways to alter product distributions by changing substrate affinities through concise mutations in binding channels. Several FAS constructs were generated ranging from first successes, where short FA were produced as side products, to FAS where native chain length programming was overwritten and only short FA were produced.
Furthermore, another engineering target was addressed with the modification of domain-domain interactions on FAS. For its exploitation to direct synthesis, contact surfaces on catalytic domains were changed to interfere with acyl carrier protein binding. This channeling of the kinetic process on the enzyme led to similar successes and short FA became the primary product.
The two approaches have proven to be potent tools to introduce systems of chain length control in FAS. This rational engineering has the big advantage that it is mostly minimally invasive and due to the high conservation of de novo FA synthesis, individual mutations could easily be used in other FAS (and their organisms) as well. Even heterologous expression of modified FAS genes is feasible.
Engineering was not only tested in a defined in vitro environment and but also in S. cerevisiae as an exemplary in vivo system. The results eventually confirmed the in vitro findings and proved that the chosen engineering could be transferred to more complex systems. Even before any optimization for highest output, the titers of short FA from S. cerevisiae fermentation matched previous reports with 118 mg/L.
In sum, this work covers several layers from basic research to preliminary applications. The presented modifications to create short FA producing FAS can be a key step in synthesis pathways and will likely enable a whole range of new succeeding research. It can be seen as a valuable contribution towards establishing novel ways for the production of chemicals from renewable sources.
Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker’s yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker’s yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker’s yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Pulsed electron-electron double resonance (PELDOR), also called Double Electron-Electron Resonance, (DEER) is a pulsed EPR technique that can provide structural information of biomolecules, such as proteins or nucleic acids, complementary to other structure determination methods by measuring long distances (from 1.5 up to 10 nm) between two paramagnetic labels. Incorporation of the rigid Ç-label pairwise into DNA or RNA molecules enables the determination not only of the distance but also of the mutual orientation between the two Ç-labels by multi-frequency orientation-selective PELDOR data (X-, Q- and G-band frequencies). Thus, information about the orientation of secondary structure elements of nucleic acids can be revealed and used as additional angular information for structure determination. Since Ç does not have motion independent from the helix where it resides, the conformational flexibility of the nucleic acid molecule can be directly determined. This thesis demonstrates the advancement of PELDOR spectroscopy, beyond its original scope of distance measurements, to determine the mutual orientation between two rigid spin labels towards the characterization of the conformational space sampled by highly flexible nucleic acid molecules. Applications of the methodology are shown on two systems: a three-way junction, namely a cocaine aptamer in its bound-state, and a two-way junction, namely a bent DNA.
More in detail, the conformational changes of the cocaine aptamer upon cocaine binding were investigated by analysis of the distance distributions. The cocaine-bound and the unbound states could be differentiated by their conformational flexibility, which decreases in the presence of the ligand. Moreover, the obtained distance distributions revealed a small change in the mean distance between the two spin labels upon cocaine binding. This indicates a ligand-induced conformational change, which presumably originates at the junction where cocaine is known to bind. The investigation of the relative orientation between the two spin-labeled helices of the aptamer revealed further structural insights into the conformational dynamics of the cocaine-bound state. The angular information from the orientation-selective PELDOR data and the a priori knowledge about the secondary structure of the aptamer were helpful in obtaining a molecular model describing its global folding and flexibility. In spite of a large flexible aptamer, the kink angle between the Ç-labeled helices was found to be rather well-defined.
As for the bent DNA molecule, a two-step protocol was proposed to investigate the conformational flexibility. In the first step, a database with all the possible conformers was created, using available restraints from NMR and distance restraints derived from PELDOR. In a second step, a weighted ensemble of these conformers fitting the multi-frequency PELDOR data was built. The uniqueness of the obtained structural ensemble was checked by validation against an independent PELDOR data set recorded at a higher magnetic field strength. In addition, the kink and twist angle pairs were determined and the resulting structural ensemble was compared with the conformational space deduced both from FRET experiments and from the structure determined by the NMR restraints alone.
Overall, this thesis underlines the potential of using PELDOR spectroscopy combined with rigid spin labels in the context of structure determination of nucleic acids in order to determine the relative orientation between two helices, the conformational flexibility and the conformational changes of nucleic acid molecules upon ligand binding.
Cancer cells, in general and especially Rhabdomyosarcoma (RMS) cells have been reported to be highly susceptible to oxidative stress. Based on this knowledge we examined whether the inhibition of the two main antioxidant defense pathways, i.e. the thioredoxin (TRX) and the glutathione (GSH) system, represents a possible new strategy to induce cell death in RMS. To do so, we combined the -glutamylcysteine synthetase (γGCL) inhibitor buthionine sulfoximine (BSO) or the cystine/glutamate antiporter (xc-) inhibitor erastin (ERA), both GSH depleting enzymes, with the thioredoxinreductase (TrxR) inhibitor auranofin (AUR) to evaluate synergistic cell death in the alveolar RMS (ARMS) cell line RH30 and the embryonal RMS (ERMS) cells RD.
Furthermore, we tried to unravel the underlying molecular mechanisms of AUR/BSO or AUR/ERA treatment in RMS cells. Thereby we showed that AUR/BSO as well as AUR/ERA treatment leads to proteasome inhibition characterized by the accumulation of ubiquitinated proteins, which is in agreement with the already published ability of AUR to inhibit proteasomeassociated deubiquitinases (DUBs) aside from TrxR. As a consequence, the protein levels of ubiquitinated short-lived proteins, like NOXA and MCL-1, increase upon treatment with AUR/BSO or AUR/ERA. Consistently, we could detect an increased binding of NOXA to MCL-1. Interestingly, not only NOXA protein levels but also mRNA levels rise upon treatment, pointing to a transcriptional regulation of pro-apoptotic NOXA through AUR/BSO or AUR/ERA combination treatment. The fact that siRNA mediated knockdown of NOXA rescues cells from combination treatment-induced cell death strengthens the role of NOXA as an important regulator of cell death induction. Apart from proteasome inhibition and subsequent NOXA accumulation, AUR cooperates with BSO or ERA to trigger BAX/BAK activation, which is needed for cell death induction, too. Additionally, loss of mitochondrial membrane potential (MMP) as well as caspase activation and PARP cleavage is detected after treatment of RMS cells with AUR/BSO or AUR/ERA.
Except of apoptotic cell death we also detected features of iron-dependent ferroptosis after treatment with AUR/BSO or AUR/ERA. This is not surprising, since BSO and ERA already have been described to induce ferroptotic cell death. Although lipid peroxidation takes place in both cell lines, only in RH30 cells, cell death seems to be partially ferroptosis-dependent, since especially in this cell line AUR/BSO- or AUR/ERA-induced cell death can be rescued with different ferroptosis inhibitors.
Although both combination treatments, AUR/BSO as well as AUR/ERA, induce production of reactive oxygen species (ROS), only the thiol-containing ROS scavengers GSH and its precursor N-acetylcysteine (NAC), but not the non-thiolcontaining antioxidant α-Tocopherol (α-Toc), consistently prevent proteasome inhibition, NOXA accumulation and cell death.
Additionally, we demonstrated that BSO and ERA abolish AUR-mediated upregulation of GSH thereby releasing the AUR cytotoxic effect on RMS cells, in line with the described ability of cysteines to inhibit the function of AUR. Together, this points to the conclusion that GSH depletion, rather than an increase in ROS levels, is important for AUR/BSO- or AUR/ERA-induced cell death.
In conclusion, through revealing that the antitumor activity of AUR is enhanced in combination with GSH depleting agents, we identified redox homeostasis as a new and promising target for the treatment of RMS cells.
Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.
The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.
Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour.
Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology.
With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential.
Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death.
Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.
Riboswitches are an important class of regulatory RNA elements that respond to cellular metabolite concentrations to regulate gene expression in a highly selective manner. 2’-deoxyguanosine-sensing (2’dG) riboswitches represent a unique riboswitch subclass only found in the bacterium Mesoplasma florum and are closely related to adenine- and guanine-sensing riboswitches. The I-A type 2’dG-sensing riboswitch represses the expression of ribonucleotide reductase genes at high cellular concentrations of 2’dG as a result of premature transcription termination.
Increasing evidence within the last decade suggests that transcriptional regulation by riboswitches is controlled kinetically and emphasizes the importance of co-transcriptional folding.2–4 Addition of single nucleotides to nascent transcripts causes a continuous shift in structural equilibrium, where refolding rates are competing with the rate of transcription.5,6
For transcriptional riboswitches, both ligand binding and structural rearrangements within the expression platform are precisely coordinated in time with the rate of transcription. The current thesis investigates the mechanistic details of transcriptional riboswitch regulation using the I-A 2’dG-sensing riboswitch as an example for a riboswitch that acts under kinetic control.
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
In dieser Dissertation wurde die Rolle des Proteins Carboxypeptidase E (CPE) im Glioblastom (GBM) untersucht. Ursprünglich wurde CPE in der neuroendokrinen Regulation beschrieben, wo es die Reifung der meisten Neuropeptide und Hormone reguliert und somit Einfluss auf Stoffwechsel und humorale Effekte hat (Fricker et al., 1982; Fricker & Snyder, 1982 and 1983; Davidson & Hutton, 1987; Shen & Loh, 1997; Lou et al., 2005). Ab 1989 wurde CPE in unterschiedlichen Tumorentitäten nachgewiesen (Grimwood et al., 1989; Manser et al., 1991), jedoch ohne Hinweise, welche Bedeutung das Protein dort haben könnte. Erst im letzten Jahrzehnt konnten sowohl pro- als auch anti-tumorigene Wirkungen von CPE gezeigt werden. Die beschriebenen Wirkungen von CPE sind jedoch von dessen Isoform abhängig. Das ∂(delta)N-trunkierte CPE zeigte sich mit erhöhtem Tumorwachstum und schlechter Überlebensprognose in verschiedenen Krebsentitäten assoziiert (Murthy et al., 2010; Lee et al., 2011; Zhou et al., 2013). Im Gegensatz dazu verringerte sezerniertes CPE (sCPE) im Fibrosarkom und Glioblastom die Zellmigration, was einen anti-tumorigenen Effekt suggeriert (Höring et al., 2012; Murthy et al., 2013a). Die Molekularmechanismen, die für die Regulation der Migration zuständig sind, sind jedoch kaum untersucht. Die meisten Untersuchungen von sCPE in Normal- und Tumorgewebe beschränken sich hauptsächlich auf Apoptose und Zellüberleben (Skalka et al., 2013; Murthy et al., 2013b; Cheng et al., 2013; Selvaraj et al., 2015; Cheng et al., 2015). Die vorliegende Arbeit ist demzufolge die erste Studie, die sich dem Mechanismus der Migrationsregulation durch sCPE im Glioblastom widmet.
Humane Gliome stellen die größte und bösartigste Gruppe hirneigener Tumore dar. Bösartige Gliome sind höchst resistent gegen alle zurzeit verfügbaren Behandlungsmethoden. Einer der Hauptgründe dafür ist, dass die Tumorzellen durch diffuse Infiltration in das Gehirn einwandern können. Ferner sind Gliomzellen metabolisch sehr aktiv und können sich dadurch an schnell verändertes Milieu anpassen (Fack et al., 2015; Demeure et al., 2016). Über die grundlegenden Mechanismen für diese Art des infiltrierenden Tumorwachstums ist bisher noch nicht viel bekannt. Zurzeit sind nur wenige Schlüsselfaktoren beschrieben, die den sogenannten Mechanismus der Migration oder Proliferation ("go or grow") in bösartigen Tumoren beeinflussen: wenige Transkriptionsfaktoren, miRNAs sowie metabolische Faktoren. Interessanterweise, sind miRNAs zum Teil mit der Regulation des Metabolismus in Tumorzellen assoziiert. Eine vorangehende Studie aus unserem Labor hat sCPE aufgrund seines Potentials, Zellwanderung zu verringern, als einen weiteren Schlüsselfaktor identifiziert. Wir konnten zeigen, dass sCPE in der Gliomzelllinie LNT-229 zur einer differentiellen Regulation von Migration und Proliferation führt (Höring et al., 2012). Die vorliegende Arbeit widmet sich nun der Frage nach den genauen zugrundeliegenden Mechanismen, wie sCPE seine Effekte auf molekularer Ebene vermittelt. Darüber hinaus soll geklärt werden, ob sCPE auch in der metabolischen Adaptation eine Rolle spielt und dadurch ebenfalls die Gliomzellmigration beeinflußen kann.
The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.