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Schlagworte
- Trypanosoma brucei (4)
- Myc (3)
- Biene (2)
- Transkription (2)
- Variant surface glycoprotein (2)
- 5' UTR (1)
- ATF4 (1)
- Apis mellifera (1)
- Bayerische Alpen <Motiv> (1)
- BioID (1)
- Breeding (1)
- C.376A>G (p.S126G) (1)
- C4da (1)
- CRISPR (1)
- CRISPR/Cas-Methode (1)
- CRISPR/Cas9 (1)
- Cancer (1)
- Cancer Metabolism (1)
- Cognition (1)
- Cognitive consistency (1)
- Cognitive profile (1)
- Colorectal Cancer (1)
- Colorectal cancer (1)
- DExD/H box protein (1)
- Degradation (1)
- Deubiquitination (1)
- Diffusion coefficient (1)
- Diffusionskoeffizient (1)
- Eclosion (1)
- Eclosion hormone (1)
- Einzelmolekülmikroskopie (1)
- Endobrachyösophagus (1)
- Epithelial lineage (1)
- Esophageal adenocarcinoma (1)
- Esophageal disease (1)
- Fabry disease (1)
- GPI-anchored protein (1)
- Gastroesophageal reflux (1)
- Genexpression (1)
- Glutamine (1)
- Heuschrecken (1)
- Honeybee (1)
- Immune evasion (1)
- Immunevasion (1)
- Immunohistochemistry (1)
- Individual differences (1)
- Intestinal metaplasia (1)
- Klimaänderung (1)
- Kognition (1)
- Learning (1)
- Lernen (1)
- Lung Cancer (1)
- Lung squamous cancer cells (1)
- Membranproteine (1)
- Metabolismus (1)
- Mevalonate Pathway (1)
- Mikroben (1)
- Mikrobiom <Genetik> (1)
- Mikroklima (1)
- Mikroorganismus (1)
- Mouse Model (1)
- N-Myc (1)
- NRF2 (1)
- NSCLC (1)
- Nahrung (1)
- Neuroendokrines System (1)
- Neuropeptide (1)
- Nicht-kleinzelliges Bronchialkarzinom (1)
- Oncoprotein (1)
- Onkoprotein (1)
- Organoid (1)
- PAF1c (1)
- PROTACs (1)
- Pause Release (1)
- Prothoracic gland (1)
- Prothoracicotropic hormone (1)
- RNA Polymerase II (RNAPII) (1)
- Regulation of expression (1)
- Resistenz (1)
- Retinoesäure (1)
- Retinoic acid (1)
- Ribosome (1)
- Ribosome biogenesis (1)
- Single-molecule fluorescence microscopy (1)
- Single-molecule tracking (1)
- Streptavidin (1)
- Taufliege (1)
- Telomer <Molekulargenetik> (1)
- Transcription Regulation (1)
- Transcription elongation (1)
- Transcriptional Stress Response (1)
- Translation <Genetik> (1)
- Translation regulation (1)
- Trypanosoma vivax (1)
- Ubiquitin (1)
- Ubiquitin Specific Protease 11 (1)
- VSG (1)
- VSG structure (1)
- Variant Surface Glycoprotein (1)
- Varroa destructor (1)
- Varroa mites (1)
- Varroa resistance (1)
- Zellskelett (1)
- Zersetzungsprozess (1)
- Zytoskelett (1)
- Züchtung (1)
- antibody fluorescence signals (1)
- biotechnical Varroa control (1)
- biotic interactions (1)
- bush ecotone (1)
- cMYC regulation (1)
- crystallization (1)
- dead organic material (1)
- decoherence (1)
- decomposition (1)
- disease model (1)
- early cosmology (1)
- elevational gradients (1)
- globotriaosylceramide (1)
- hedge index (1)
- hedgerow (1)
- heritability (1)
- honeybees (1)
- immunofluorescence detection (1)
- in vivo (1)
- in vivo genome editing (1)
- induced pluripotent stem cells (1)
- melanoma (1)
- messenger RNA (1)
- messenger RNA regulation (1)
- microbes (1)
- mite non-reproduction (1)
- modified inflation (1)
- plant-herbivore-interactions (1)
- protein folding (1)
- protein localization (1)
- qubit (1)
- qubit interaction (1)
- rRNA processing (1)
- recapping (1)
- selection (1)
- semi-natural habitat (1)
- sensory neurons (1)
- succession (1)
- telomere-associated protein (1)
- thiol starvation (1)
- transcription elongation factor A (SII)-like 1 (TCEAL1) (1)
- transitional shrubland (1)
- true bug (1)
- variants of unknown significance (1)
- v’Td (1)
- α-Galactosidase A (1)
Institut
- Theodor-Boveri-Institut für Biowissenschaften (22) (entfernen)
Sonstige beteiligte Institutionen
African trypanosomes are unicellular parasites that cause nagana and sleeping sickness in livestock and man, respectively. The major pathogens for the animal disease include Trypanosoma vivax, T. congolense, and T. brucei brucei, whereas T. b. gambiense and T. b. rhodesiense are responsible for human infections. Given that the bloodstream form (BSF) of African trypanosomes is exclusively extracellular, its cell surface forms a critical boundary with the host environment. The cell surface of the BSF African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). This surface protein acts as an impenetrable shield that protects the cells from host immune factors and is also involved in antibody clearance and antigenic variation, which collectively ensure that the parasite stays ahead of the host immune system. Gene expression in T. brucei is markedly different from other eukaryotes: most genes are transcribed as long polycistronic units, processed by trans-splicing a 39-nucleotide mini exon at the 5′ and polyadenylation at the 3′ ends of individual genes to generate the mature mRNA.
Therefore, gene expression in T. brucei is regulated post-transcriptionally, mainly by the action of RNA binding proteins (RBPs) and conserved elements in the 3′ untranslated regions (UTR) of transcripts. The expression of VSGs is highly regulated, and only a single VSG gene is expressed at a time from one of the ~15 subtelomeric domains termed bloodstream expression sites (BES). When cells are engineered to simultaneously express two VSGs, the total VSG mRNA do not exceed the wild type amounts. This suggests that a robust VSG mRNA balancing mechanism exists in T. brucei. The present study uses inducible and constitutive expression of ectopic VSG genes to show that the endogenous VSG mRNA is regulated only if the second VSG is properly targeted to the ER. Additionally, the endogenous VSG mRNA response is triggered when high amounts of the GFP reporter with a VSG 3′UTR is targeted to the ER. Further evidence that non-VSG ER import signals can efficiently target VSGs to the ER is presented. This study suggests that a robust trans-regulation of the VSG mRNA is elicited at the ER through a feedback loop to keep the VSG transcripts in check and avoid overshooting the secretory pathway capacity.
Further, it was shown that induction of expression of the T. vivax VSG ILDat1.2 in T. brucei causes a dual cell cycle arrest, with concomitant upregulation of the protein associated with differentiation (PAD1) expression. It could be shown that T. vivax VSG ILDat1.2 can only be sufficiently expressed in T. brucei after replacing its native GPI signal peptide with that of a T. brucei VSG. Taken together, these data indicate that inefficient VSG GPI anchoring and expression of low levels of the VSG protein can trigger differentiation from slender BSF to stumpy forms. However, a second T. vivax VSG, ILDat2.1, is not expressed in T. brucei even after similar modifications to its GPI signals. An X-ray crystallography approach was utilized to solve the N-terminal domain (NTD) structure of VSG ILDat1.2. This is first structure of a non-T. brucei VSG, and the first of a surface protein of T. vivax to be solved. VSG ILDat1.2 NTD maintains the three-helical bundle scaffold conserved in T. brucei surface proteins. However, it is likely that there are variations in the architecture of the membrane proximal region of the ILDat1.2 NTD and its CTD from T. brucei VSGs. The tractable T. brucei system is presented as a model that can be used to study surface proteins of related trypanosome species, thus creating avenues for further characterization of trypanosome surface coats.
The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; Braf\(^{CA}\); Pten\(^{lox/+}\) melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.
Chapter I: Introduction
Temperature is a major driver of biodiversity and abundance patterns on our planet, which becomes particularly relevant facing the entanglement of an imminent biodiversity and climate crisis. Climate shapes the composition of species assemblages either directly via abiotic filtering mechanisms or indirectly through alterations in biotic interactions. Insects - integral elements of Earth’s ecosystems - are affected by climatic variation such as warming, yet responses vary among species. While species’ traits, antagonistic biotic interactions, and even species’ microbial mutualists may determine temperature-dependent assembly processes, the lion’s share of these complex relationships remains poorly understood due to methodological constraints. Mountains, recognized as hotspots of diversity and threatened by rapidly changing climatic conditions, can serve as natural experimental settings to study the response of insect assemblages and their trophic interactions to temperature variation, instrumentalizing the high regional heterogeneity of micro- and macroclimate. With this thesis, we aim to enhance our mechanistic understanding of temperature-driven assembly processes within insect communities, exemplified by Orthoptera, that are significant herbivores in temperate mountain grassland ecosystems. Therefore, we combined field surveys of Orthoptera assemblages on grassland sites with molecular tools for foodweb reconstruction, primarily leveraging the elevational gradients offered by the complex topography within the Berchtesgaden Alpine region (Bavaria, Germany) as surrogate for temperature variation (space-for-time substitution approach). In this framework, we studied the effects of temperature variation on (1) species richness, abundance, community composition, and interspecific as well as intraspecific trait patterns, (2) ecological feeding specialisation, and (3) previously neglected links to microbial associates found in the faeces.
Chapter II: Temperature-driven assembly processes
Climate varies at multiple scales. Since microclimate is often overlooked, we assessed effects of local temperature deviations on species and trait compositions of insect communities along macroclimatic temperature gradients in Chapter II. Therefore, we employed joint species distribution modelling to explore how traits drive variation in the climatic niches of Orthoptera species at grassland sites characterized by contrasting micro- and macroclimatic conditions. Our findings revealed two key insights: (1) additive effects of micro- and macroclimate on the diversity, but (2) interactive effects on the abundance of several species, resulting in turnover and indicating that species possess narrower climatic niches than their elevational distributions might imply. This chapter suggests positive effects of warming on Orthoptera, but also highlights that the interplay of macro- and microclimate plays a pivotal role in structuring insect communities. Thus, it underscores the importance of considering both elements when predicting the responses of species to climate change. Additionally, this chapter revealed inter- and intraspecific effects of traits on the niches and distribution of species.
Chapter III: Dietary specialisation along climatic gradients
A crucial trait linked to the position of climatic niches is dietary specialisation. According to the ‘altitudinal niche-breadth hypothesis’, species of high-elevation habitats should be less specialized compared to their low-elevation counterparts. However, empirical evidence on shifts in specialization is scarce for generalist insect herbivores and existing studies often fail to control for the phylogeny and abundance of interaction partners. In Chapter III, we used a combination of field observations and amplicon sequencing to reconstruct dietary relationships between Orthoptera and plants along an extensive temperature gradient. We did not find close but flexible links between individual grasshopper and plant taxa in space. While interaction network specialisation increased with temperature, the corrected dietary specialisation pattern peaked at intermediate elevations on assemblage level. These nuanced findings demonstrate that (1) resource availability, (2) phylogenetic relationships, and (3) climate can affect empirical foodwebs intra- and interspecifically and, hence, the dietary specialisation of herbivorous insects. In this context, we discuss that the underlying mechanisms involved in shaping the specialisation of herbivore assemblages may switch along temperature clines.
Chapter IV: Links between faecal microbe communities, feeding habits, and climate
Since gut microbes affect the fitness and digestion of insects, studying their diversity could provide novel insights into specialisation patterns. However, their association with insect hosts that differ in feeding habits and specialisation has never been investigated along elevational climatic gradients. In Chapter IV, we utilized the dietary information gathered in Chapter III to characterize links between insects with distinct feeding behaviour and the microbial communities present in their faeces, using amplicon sequencing. Both, feeding and climate affected the bacterial communities. However, the large overlap of microbes at site level suggests that common bacteria are acquired from the shared feeding environment, such as the plants consumed by the insects. These findings emphasize the influence of a broader environmental context on the composition of insect gut microbial communities.
Chapter V: Discussion & Conclusions
Cumulatively, the sections of this dissertation provide support for the hypothesis that climatic conditions play a role in shaping plant–herbivore systems. The detected variation of taxonomic and functional compositions contributes to our understanding of assembly processes and resulting diversity patterns within Orthoptera communities, shedding light on the mechanisms that structure their trophic interactions in diverse climates. The combined results presented suggest that a warmer climate could foster an increase of Orthoptera species richness in Central European semi-natural grasslands, also because the weak links observed between insect herbivores and plants are unlikely to limit decoupled range shifts. However, the restructuring of Orthoptera communities in response to warmer temperatures depends on species' traits such as moisture preferences or phenology. Notably, we were able to demonstrate a crucial role of microclimate for many species, partly unravelling narrower climatic niches than their elevational ranges suggest. We found evidence that not only Orthoptera community composition, specialisation, and traits varied along elevational gradients, but even microbial communities in the faeces of Orthoptera changed, which is a novel finding. This complex restructuring and reassembly of communities, coupled with the nonlinear specialisation of trophic interactions and a high diversity of associated bacteria, emphasize our currently incomplete comprehension of how ecosystems will develop under future climatic conditions, demanding caution in making simplified predictions for biodiversity change under climate warming. Since these predictions may benefit from including biotic interactions and both, micro- and macroclimate based on our findings, conservation authorities and practitioners must not neglect improving microclimatic conditions to ensure local survival of a diverse set of threatened and demanding species. In this context, mountains can play a pivotal role for biodiversity conservation since these offer heterogeneous microclimatic conditions in proximity that can be utilized by species with distinct niches.
The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc’s structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel–Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field.
In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines.
In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs.
In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs.
Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy.
Structure and dynamics of the plasma membrane: a single-molecule study in \(Trypanosoma\) \(brucei\)
(2024)
The unicellular, flagellated parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in livestock. In the last decades, it has become an established eukaryotic model organism in the field of biology, as well as in the interdisciplinary field of biophysics. For instance, the dense variant surface glycoprotein (VSG) coat offers the possibility to study the dynamics of GPI-anchored proteins in the plasma membrane of living cells. The fluidity of the VSG coat is not only an interesting object of study for its own sake, but is critically important for the survival of the parasite in the mammalian host. In order to maintain the integrity of the coat, the entire VSG coat is recycled within a few minutes. This is surprisingly fast for a purely diffusive process with the flagellar pocket (FP) as the sole site for endo- and exocytosis. Previous studies characterising VSG dynamics using FRAP reported diffusion coefficients that were not sufficient to to enable fast turnover based on passive VSG randomisation on the trypanosome surface.
In this thesis, live-cell single-molecule fluorescence microscopy (SMFM) was employed to elucidate whether VSG diffusion coefficients were priorly underestimated or whether directed forces could be involved to bias VSGs towards the entrance of the FP. Embedding the highly motile trypanosomes in thermo-stable hydrogels facilitated the investigation of VSG dynamics on living trypanosomes at the mammalian host's temperature of 37°C. To allow for a spatial correlation of the VSG dynamics to the FP entrance, a cell line was employed harbouring a fluorescently labelled structure as a reference. Sequential two-colour SMFM was then established to allow for recording and registration of the dynamic and static single-molecule information.
In order to characterise VSG dynamics, an algorithm to obtain reliable information from short trajectories was adapted (shortTrAn). It allowed for the quantification of the local dynamics in two distinct scenarios: diffusion and directed motion. The adaptation of the algorithm to the VSG data sets required the introduction of an additional projection filter. The algorithm was further extended to take into account the localisation errors inherent to single-particle tracking. The results of the quantification of diffusion and directed motion were presented in maps of the trypanosome surface, including an outline generated from a super-resolved static structure as a reference. Information on diffusion was displayed in one map, an ellipse plot. The colour code represented the local diffusion coefficient, while the shape of the ellipses provided an indication of the diffusion behaviour (aniso- or isotropic diffusion). The eccentricity of the ellipses was used to quantify deviations from isotropic diffusion. Information on directed motion was shown in three maps: A velocity map, representing the amplitude of the local velocities in a colour code. A quiver plot, illustrating the orientation of directed motion, and a third map which indicated the relative standard error of the local velocities colour-coded. Finally, a guideline based on random walk simulations was used to identify which of the two motion scenarios dominated locally. Application of the guideline to the VSG dynamics analysed by shortTrAn yielded supermaps that showed the locally dominant motion mode colour-coded.
I found that VSG dynamics are dominated by diffusion, but several times faster than previously determined. The diffusion behaviour was additionally characterised by spatial heterogeneity. Moreover, isolated regions exhibiting the characteristics of round and elongated traps were observed on the cell surface. Additionally, VSG dynamics were studied with respect to the entrance of the FP. VSG dynamics in this region displayed similar characteristics compared to the remainder of the cell surface and forces biasing VSGs into the FP were not found.
Furthermore, I investigated a potential interference of the attachment of the cytoskeleton to the plasma membrane with the dynamics of VSGs which are anchored to the outer leaflet of the membrane. Preliminary experiments were conducted on osmotically swollen trypanosomes and trypanosomes depleted for a microtubule-associated protein anchoring the subpellicular microtubule cytoskeleton to the plasma membrane. The measurements revealed a trend that detachment of the cytoskeleton could be associated with a reduction in the VSG diffusion coefficient and a loss of elongated traps. The latter could be an indication that these isolated regions were caused by underlying structures associated with the cytoskeleton.
The measurements on cells with an intact cytoskeleton were complemented by random walk simulations of VSG dynamics with the newly determined diffusion coefficient on long time scales not accessible in experiments. Simulations showed that passive VSG randomisation is fast enough to allow for a turnover of the full VSG coat within a few minutes. According to an estimate based on the known rate of endocytosis and the newly determined VSG diffusion coefficient, the majority of exocytosed VSGs could escape from the FP to the cell surface without being immediately re-endocytosed.
Colon carcinomas (CRC) are statistically among the most fatal cancer types and hence one of the top reasons for premature mortality in the developed world. CRC cells are characterized by high proliferation rates caused by deregulation of gene transcription of proto-oncogenes and general chromosomal instability. On macroscopic level, CRC cells show a strongly altered nutrient and energy metabolism.
This work presents research to understand general links between the metabolism and transcription alteration. Mainly focussing on glutamine dependency, shown in colon carcinoma cells and expression pathways of the pro-proliferation protein c-MYC.
Previous studies showed that a depletion of glutamine in the cultivation medium of colon carcinoma cell lines caused a proliferation arrest and a strong decrease of overall c-MYC levels. Re-addition of glutamine quickly replenished c-MYC levels through an unknown mechanism. Several proteins altering this regulation mechanism were identified and proposed as possible starting point for further in detail studies to unveil the precise biochemical pathway controlling c-MYC translation repression and reactivation in a rapid manner.
On a transcriptional level the formation of RNA:DNA hybrids, so called R-loops, was observed under glutamine depleted conditions. The introduction and overexpression of RNaseH1, a R-loop degrading enzyme, in combination with an ectopically expressed c-MYC variant, independent of cellular regulation mechanisms by deleting the regulatory 3’-UTR of the c-MYC gene, lead to a high rate of apoptotic cells in culture. Expression of a functionally inactive variant of RNaseH1 abolished this effect. This indicates a regulatory function of R-loops formed during glutamine starvation in the presence of c-MYC protein in a cell. Degradation of R-loops and high c-MYC levels in this stress condition had no imminent effect on the cell cycle progression is CRC cells but disturbed the nucleotide metabolism. Nucleotide triphosphates were strongly reduced in comparison to starving cells without R-loop degradation and proliferating cells.
This study proposes a model of a terminal cycle of transcription termination, unregulated initiation and elongation of transcription leading to a depletion of energy resources of cells. This could finally lead to high apoptosis of the cells. Sequencing experiments to determine a coinciding of termination sites and R-loop formation sides failed so far but show a starting point for further studies in this essential survival mechanism involving R-loop formation and c-MYC downregulation.
The variant surface glycoprotein (VSG) of African trypanosomes plays an essential role in protecting the parasites from host immune factors. These trypanosomes undergo antigenic variation resulting in the expression of a single VSG isoform out of a repertoire of around 2000 genes. The molecular mechanism central to the expression and regulation of the VSG is however not fully understood.
Gene expression in trypanosomes is unusual due to the absence of typical RNA polymerase II promoters and the polycistronic transcription of genes. The regulation of gene expression is therefore mainly post-transcriptional. Regulatory sequences, mostly present in the 3´ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 100 % conserved 16mer motif within the 3´ UTR has been shown to modulate the stability of VSG transcripts and hence their expression. As a stability-associated sequence element, the absence of nucleotide substitutions in the motif is however unusual. It was therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts.
In this study, it was demonstrated that the 100 % conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. It was further shown that the intact motif in the active VSG 3´ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Crosstalk between the VSG and procyclin genes during differentiation to the insect vector stage is also unaffected in cells with a mutated 16mer motif. Ectopic overexpression of a second VSG however requires the intact motif to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The 16mer motif therefore plays a dual role in VSG in situ switching and stability of VSG transcripts. The additional role of the 16mer in the essential process of antigenic variation appears to be the driving force for the 100 % conservation of this RNA motif.
A screen aimed at identifying candidate RNA-binding proteins interacting with the 16mer motif, led to the identification of a DExD/H box protein, Hel66. Although the protein did not appear to have a direct link to the 16mer regulation of VSG expression, the DExD/H family of proteins are important players in the process of ribosome biogenesis. This process is relatively understudied in trypanosomes and so this candidate was singled out for detailed characterisation, given that the 16mer story had reached a natural end point. Ribosome biogenesis is a major cellular process in eukaryotes involving ribosomal RNA, ribosomal proteins and several non-ribosomal trans-acting protein factors. The DExD/H box proteins are the most important trans-acting protein factors involved in the biosynthesis of ribosomes. Several DExD/H box proteins have been directly implicated in this process in yeast. In trypanosomes, very few of this family of proteins have been characterised and therefore little is known about the specific roles they play in RNA metabolism. Here, it was shown that Hel66 is involved in rRNA processing during ribosome biogenesis. Hel66 localises to the nucleolus and depleting the protein led to a severe growth defect. Loss of the protein also resulted in a reduced rate of global translation and accumulation of rRNA processing intermediates of both the small and large ribosomal subunits. Hel66 is therefore an essential nucleolar DExD/H protein involved in rRNA processing during ribosome biogenesis. As very few protein factors involved in the processing of rRNAs have been described in trypanosomes, this finding represents an important platform for future investigation of this topic.
The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment.
De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis.
SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis.
SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation.
It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth.
A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation.
In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients.
The expression of the MYC proto-oncogene is elevated in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC). Previous findings in PDAC have shown that this increased MYC expression mediates immune evasion and promotes S-phase progression. How these functions are mediated and whether a downstream factor of MYC mediates these functions has remained elusive. Recent studies identifying the MYC interactome revealed a complex network of interaction partners, highlighting the need to identify the oncogenic pathway of MYC in an unbiased manner.
In this work, we have shown that MYC ensures genomic stability during S-phase and prevents transcription-replication conflicts. Depletion of MYC and inhibition of ATR kinase showed a synergistic effect to induce DNA damage. A targeted siRNA screen targeting downstream factors of MYC revealed that PAF1c is required for DNA repair and S-phase progression. Recruitment of PAF1c to RNAPII was shown to be MYC dependent. PAF1c was shown to be largely dispensable for cell proliferation and regulation of MYC target genes.
Depletion of CTR9, a subunit of PAF1c, caused strong tumor regression in a pancreatic ductal adenocarcinoma model, with long-term survival in a subset of mice. This effect was not due to induction of DNA damage, but to restoration of tumor immune surveillance.
Depletion of PAF1c resulted in the release of RNAPII with transcription elongation factors, including SPT6, from the bodies of long genes, promoting full-length transcription of short genes. This resulted in the downregulation of long DNA repair genes and the concomitant upregulation of short genes, including MHC class I genes. These data demonstrate that a balance between long and short gene transcription is essential for tumor progression and that interference with PAF1c levels shifts this balance toward a tumor-suppressive transcriptional program. It also directly links MYC-mediated S-phase progression to immune evasion. Unlike MYC, PAF1c has a stable, known folded structure; therefore, the development of a small molecule targeting PAF1c may disrupt the immune evasive function of MYC while sparing its physiological functions in cellular growth.
Gastroesophageal junction (GEJ), demarcating the region where the distal esophagus meets with the proximal stomach region, is known for developing pathological conditions, including metaplasia and esophageal adenocarcinoma (EAC). It is essential to understand the mechanisms of developmental stages which lead to EAC since the incidence rate of EAC increased over 7-fold during the past four decades, and the overall five years survival rate is 18.4%. In most cases, patients are diagnosed in the advanced stage without prior symptoms. The main precursor for the development of EAC is a pre-malignant condition called Barrett's esophagus (BE). BE is the metaplastic condition where the multilayered squamous epithelium of the native esophagus is replaced by specialized single-layered columnar epithelium, which shows the molecular characteristics of the gastric as well as intestinal epithelium. The main risk factors for BE development include chronic gastro-esophageal acid reflux disease (GERD), altered microbiota, and altered retinoic acid signaling (RA). The cell of origin of BE is under debate due to a lack of clear evidence demonstrating the process of BE initiation. Here, I investigated how GEJ homeostasis is maintained in healthy tissue by stem cell regulatory morphogens, the role of vitamin A (RA signaling), and how its alteration contributes to BE development.
In the first part of my thesis, I showed the presence of two types of epithelial cells, the squamous type in the esophagus and the columnar type in the stomach region in the GEJ, using single-molecule RNA in situ hybridization (smRNA-ISH) and immunohistochemistry. Employing lineage tracing in the mouse model, I have demonstrated that the esophageal epithelial and stomach epithelial cells derived from two distinct epithelial stem cell lineages in the GEJ. The border between squamous and columnar epithelial cells in the Squamo-columnar junction (SCJ) of GEJ is regulated by opposing Wnt microenvironments. The regeneration of stomach columnar epithelial stem cells is maintained by Wnt activating signal from the stromal compartment while squamous epithelial stem cells of the esophagus are maintained by the Wnt inhibitory signals. I recapitulated the in vivo GEJ epithelial stem cell maintenance by using in vitro epithelial 3D organoid culture model. The growth and propagation of stomach columnar epithelial organoids depend on Wnt growth factors, while squamous epithelial organoids' development needs Wnt-deficient culture conditions.
Further, single-cell RNA sequence (scRNA-seq) analysis of organoid-derived epithelial cells revealed the non-canonical Wnt/ planar cell polarity (PCP) pathway involvement in regulating the squamous epithelial cells. In contrast, columnar stomach epithelial cells are regulated by the canonical Wnt/ beta-catenin and non-canonical Wnt/Ca2+ pathways. My data indicate that the SCJ epithelial cells that merge at the GEJ are regulated by opposing stromal Wnt factors and distinct Wnt pathway signaling in the epithelial cells.
In the second part of the thesis, I investigated the role of Vitamin A-derived bioactive compound RA on esophageal and stomach epithelial stem cells. In vitro treatment of esophageal and stomach, epithelial organoids with RA or its pharmacological inhibitor BMS 493 revealed that each cell type was regulated distinctly. I observed that enhanced RA promoted esophageal stem cell differentiation and loss of stratification, while RA inhibition led to enhanced stemness and regeneration of the esophagus stratified epithelium. As opposed to the esophagus, RA signaling is active in the stomach organoids, and inhibition of RA reduces the growth of stomach organoids. Global transcriptomic data and scRNA-seq data revealed that RA signaling induces dormancy phenotype in the esophageal cells. In contrast, the absence of RA in stomach epithelial cells induces the expression of genes associated with BE. Thus, spatially defined regulation of Wnt and RA signaling at GEJ is critical for healthy homeostasis, and its perturbation leads to disease development.