Chemische Biologie

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Design principles and applications of synthetic self-replicating RNAs
(2023-06-01) Wagner, Alexander; Mutschler, Hannes
With the advent of ever more sophisticated methods for the in vitro synthesis and the in vivo delivery of RNAs, synthetic mRNAs have gained substantial interest both for medical applications, as well as for biotechnology. However, in most biological systems exogeneous mRNAs possess only a limited half-life, especially in fast dividing cells. In contrast, viral RNAs can extend their lifetime by actively replicating inside their host. As such they may serve as scaffolds for the design of synthetic self-replicating RNAs (srRNA), which can be used to increase both the half-life and intracellular concentration of coding RNAs. Synthetic srRNAs may be used to enhance recombinant protein expression or induce the reprogramming of differentiated cells into pluripotent stem cells but also to create cell-free systems for research based on experimental evolution. In this article, we discuss the applications and design principles of srRNAs used for cellular reprogramming, mRNA-based vaccines and tools for synthetic biology. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution
Targeting the RNA-binding proteins LIN28, METTL16 and YTHDF2 using small molecules and bifunctional molecules
(2024) Goebel, Georg Lennart; Waldmann, Herbert; Mutschler, Hannes
Die Interaktion von RNA-bindenden Proteinen (RBP) und RNA spielt eine entscheidende Rolle in vielen zellulären Prozessen, deren Fehlregulation zu zahlreichen Krankheiten führen kann. Die Entdeckung von small molecules, die diese Interaktionen inhibieren, ist eine vielversprechende Strategie für neue chemische Werkzeuge und Therapeutika. Trotz einiger Berichte über small-molecule Inhibitoren gibt es noch Verbesserungsbedarf hinsichtlich Potenz, Selektivität und Zytotoxizität. In dieser Arbeit wurden small-molecule Inhibitoren für drei verschiedene RBP synthetisiert: Das onkogene miRNA-bindende Protein LIN28, die tumorfördernde Methyltransferase METTL16 und das RNA-destabilisierende Protein YTHDF2. LIN28 reguliert die Reifung von let-7 miRNAs, die die Translation onkogener Proteine unterdrücken. Seine Überexpression macht es zu einem Haupttreiber der Tumorprogression. Existierende LIN28-Inhibitoren sind unzureichend potent, daher wurden in dieser Arbeit versucht durch Scaffold- und Screening-basierte Ansätze neue Inhibitoren mit verbesserter Potenz zu identifizieren. Ein Tetrahydroquinolin-Analogon und Pyrrolinon-basierte Verbindungen zeigten dabei vielversprechende Effekte auf die let-7-Reifung und die gefundenen small molecules bilden eine wertvolle Grundlage für die Entwicklung von LIN28-addressierenden chemischen Werkzeugen und neuartigen Krebstherapeutika. METTL16 ist an der epitranskriptomischen m6A-Methylierung beteiligt und spielt eine tumorfördernde Rolle, indem es mit anderen RBP interagiert. Da bisher keine METTL16-Inhibitoren bekannt waren, wurden in dieser Arbeit die ersten Inhibitoren durch ein FP-basiertes Screening identifiziert. Eine nachfolgende strukturelle Optimierung führte zu Inhibitoren mit mikromolarer Aktivität, die wertvolle Einblicke in die Funktion von METTL16 liefern. Das m6A-erkennende Protein YTHDF2 wird mit Prozessen wie Apoptose, Zellzyklus und Metastasierung in Verbindung gebracht. Um YTHDF2 zu inhibieren, wurden zwei Ansätze verfolgt: pyrimidin-basierte PROTACs zur Degradation und small-molecule Inhibitoren, wobei optimierte Phenylpyrazole als vielversprechende YTHDF2-Inhibitoren identifiziert wurden. Die strukturelle Optimierung des ursprünglichen Treffers ergab ein 1-Naphthoyl enthaltendes Analogon, das eine potente anti-YTHDF2-Aktivität zeigte und eine potenziell fundamentale Struktur für die weitere Entwicklung von small molecules zur Inhibierung von YTHDF2 für biologische und therapeutische Anwendungen darstellt. Insgesamt konnten die Ergebnisse dieser Arbeit die Wirksamkeit der Inhibition therapeutisch relevanter RBP mittels verschiedener Strategien basierend auf small molecules demonstrieren. Während Scaffold-basierte Ansätze sich auf die Optimierung gegebener RBP-Inhibitoren konzentrierten, führten Screening-basierte Bemühungen zur Identifizierung neuer chemischer Modalitäten. Die identifizierten Verbindungen sowie die in dieser Arbeit etablierten Assays werden wertvoll für die zukünftige Entwicklung von small molecules zur Aufklärung und Modulation von RBP sein.
Identification and characterization of small-molecule modulators that promote cancer cell elimination
(2024) Binici, Aylin; Waldmann, Herbert; Watzl, Carsten
In this doctoral thesis, two strategies were used to fight cancer. The first strategy aimed to eliminate cancer via natural killer (NK) cells. Within the tumor microenvironment (TME), cancer cells and the cancer-supporting tissue hijack immunosuppressive pathways, leading to the increased secretion of immune inhibitory factors that aid tumor progression, ultimately leading to the evasion of NK-cell mediated eradication of cancer cells. A phenotypic co-culture assay was optimized to mimic the TME. The assay consists of a human lung adenocarcinoma cells A549, primary lymphocytes, and immunomodulatory factors. This setup was used to screen a library of 29,502 small molecules in search of compounds that can restore NK cell-mediated cancer cell elimination. The second strategy was focused on eliminating cancer through indirect targeting of the small GTPase Kirsten rat sarcoma (KRas). KRas is mutated in approximately 17 % of all solid tumors, and current FDA-approved drugs solely target its G12C mutant. The activity of this oncoprotein depends on its localization to the plasma membrane, and within the cell, its translocation within the cell is facilitated by its interaction with the prenyl group-binding chaperone retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta (PDEδ). Therefore, instead of directly targeting KRas, the covalent inhibitor Deltacovalin was designed to target PDEδ.
Trends in the diversification of the detergentome
(2023-09-05) Wycisk, Virginia; Wagner, Marc-Christian; Urner, Leonhard H.
Detergents are amphiphilic molecules that serve as enabling steps for today's world applications. The increasing diversity of the detergentome is key to applications enabled by detergent science. Regardless of the application, the optimal design of detergents is determined empirically, which leads to failed preparations, and raising costs. To facilitate project planning, here we review synthesis strategies that drive the diversification of the detergentome. Synthesis strategies relevant for industrial and academic applications include linear, modular, combinatorial, bio-based, and metric-assisted detergent synthesis. Scopes and limitations of individual synthesis strategies in context with industrial product development and academic research are discussed. Furthermore, when designing detergents, the selection of molecular building blocks, i. e., head, linker, tail, is as important as the employed synthesis strategy. To facilitate the design of safe-to-use and tailor-made detergents, we provide an overview of established head, linker, and tail groups and highlight selected scopes and limitations for applications. It becomes apparent that most recent contributions to the increasing chemical diversity of detergent building blocks originate from the development of detergents for membrane protein studies. The overview of synthesis strategies and molecular blocks will bring us closer to the ability to predictably design and synthesize optimal detergents for challenging future applications.
Investigation into the specificities of deubiquitinases and ubiquitin-like proteases
(2024) Zhao, Zhou; Gersch, Malte; Waldmann, Herbert
Ubiquitin as a macromolecule-based modification has been validated as the regulator of protein stability. Ubiquitin signals can be significantly diversified by forming polymerized chains as well as conjugating with some structurally similar modifiers (Ubiquitin-like proteins, Ubls). Therefore, cellular events can be modulated in a more complicated way through these modifications, which are reversible by specialized proteases called deubiquitinases (DUBs) and Ubiquitin-like proteases (ULPs). Some of these enzymes show specificity toward different modifiers, while some prefer to cleave linkage-specific polyubiquitin chains. To study the modifier specificity and linkage specificity, high quality chemical biology tools are the cornerstones. Isopeptide-linked fluorescence polarization substrates are powerful tools for analyzing modifier specificity of DUBs/ULPs quantitatively. Since Ub/Ubl-based substrates can be sensitive to harsh reaction and purification conditions, a native semisynthetic method was developed based on recombinantly expressed proteins which were further functionalized with fluorophores and purified in aqueous buffer to ensure the homogeneity of substrates. Six substrates were prepared to assemble an assay panel for characterizing several DUBs/ULPs. USP16 and USP36 were unprecedently identified as the first proteases with triple modifier specificity. Studies on linkage specificity were enabled by the development of novel diUb probes with internal warheads, which are based on alkyl bromide. Alkyl-bromide-based probes were successfully used for studying SnVTD, which is a Lys6-specific DUB. The complex structure was solved to reveal a unique recognition and activation mechanism. The probes were also applied for capturing two linkage-specific E3s. Collectively, a high-quality fluorescence polarization assay platform was established and used for exploring modifier specificity of DUBs/ULPs. Novel diUb probes were developed to capture DUBs with linkage specificity. These tools will facilitate the understanding of DUBs/ULPs.
Ribozyme-mediated RNA synthesis and replication in a model Hadean microenvironment
(2023-03-17) Salditt, Annalena; Karr, Leonie; Salibi, Elia; Le Vay, Kristian; Braun, Dieter; Mutschler, Hannes
Enzyme-catalyzed replication of nucleic acid sequences is a prerequisite for the survival and evolution of biological entities. Before the advent of protein synthesis, genetic information was most likely stored in and replicated by RNA. However, experimental systems for sustained RNA-dependent RNA-replication are difficult to realise, in part due to the high thermodynamic stability of duplex products and the low chemical stability of catalytic RNAs. Using a derivative of a group I intron as a model for an RNA replicase, we show that heated air-water interfaces that are exposed to a plausible CO2-rich atmosphere enable sense and antisense RNA replication as well as template-dependent synthesis and catalysis of a functional ribozyme in a one-pot reaction. Both reactions are driven by autonomous oscillations in salt concentrations and pH, resulting from precipitation of acidified dew droplets, which transiently destabilise RNA duplexes. Our results suggest that an abundant Hadean microenvironment may have promoted both replication and synthesis of functional RNAs.
Periodic temperature changes drive the proliferation of self-replicating RNAs in vesicle populations
(2023-03-03) Salibi, Elia; Peter, Benedikt; Schwille, Petra; Mutschler, Hannes
Growth and division of biological cells are based on the complex orchestration of spatiotemporally controlled reactions driven by highly evolved proteins. In contrast, it remains unknown how their primordial predecessors could achieve a stable inheritance of cytosolic components before the advent of translation. An attractive scenario assumes that periodic changes of environmental conditions acted as pacemakers for the proliferation of early protocells. Using catalytic RNA (ribozymes) as models for primitive biocatalytic molecules, we demonstrate that the repeated freezing and thawing of aqueous solutions enables the assembly of active ribozymes from inactive precursors encapsulated in separate lipid vesicle populations. Furthermore, we show that encapsulated ribozyme replicators can overcome freezing-induced content loss and successive dilution by freeze-thaw driven propagation in feedstock vesicles. Thus, cyclic freezing and melting of aqueous solvents – a plausible physicochemical driver likely present on early Earth – provides a simple scenario that uncouples compartment growth and division from RNA self-replication, while maintaining the propagation of these replicators inside new vesicle populations.
Towards the design and construction of self-replicating RNA nanostructures
(2024) Rubert, David; Mutschler, Hannes; Summerer, Daniel
The transition from an RNA-based world to DNA as the primary genetic material is a pivotal topic in origin-of-life research. This study aims to prototype a hybrid genome system combining DNA and RNA, capable of self-replication and evolution, initially in vitro and potentially in vivo. Using Qß replicase, segmented RNA genomes were synthesized, incorporating the Phi29 phage pRNA for self-assembly into nanorings. The replication efficacy of these replicons was evaluated both independently and in coupled assembly-replication reactions, with Qß chosen for its high amplification efficiency and template versatility. To mitigate the challenge of parasite RNA formation, which depletes essential replication components, a water-emulsion system was employed. The modular assembly of replicons into nanorings was further investigated, with a focus on system reproducibility and the effect of Mg2+ concentrations on nanoring stability. The final goal is to integrate self-replication with self-assembly, thereby creating a segmented, modular RNA genome for in vitro genetic information storage. This work addresses key challenges and advancements in developing synthetic self-replication systems for eventual genomic transplantation in E. coli.
The role of cell cycle, growth, and metabolism in species-specific differentiation timing
(2024) Schröder, Julia; Bastiaens, Philippe; Pfander, Boris
Different mammalian species progress through similar stages during embryonic development and adult life but the pace of these transitions is species-specific. While classically, developmental timing was viewed merely as a consequence of varying body sizes between species, the use of pluripotent stem cells (PSCs) has shown that species-specific developmental timing is maintained during in vitro differentiation. Since then, uncovering cell-intrinsic mechanisms that regulate the timescales of development has become a rising topic of interest. This project aimed to identify such cell-intrinsic mechanisms using in vitro neural progenitor differentiation of mouse, monkey and human PSCs. To facilitate inter-species comparisons, all cells were cultured and differentiated under harmonized conditions. Under these circumstances, mouse cells differentiated more than twice as fast as human cells, recapitulating species-specific differentiation timing. As differentiation and growth need to be tightly coordinated during development, I compared cell cycle durations across the species. Cell cycle durations followed a species-specific trend, with the human cell cycle being 1.47-fold and the monkey cell cycle 1.42- fold longer than the mouse cell cycle. To test if differentiation depends on proliferation, I performed cell cycle and growth manipulations by either a retinoblastoma knock-out line or inhibiting the mammalian target of rapamycin (mTOR). Strikingly, mTOR inhibition caused a drastic extension of cell cycle durations, yet single-cell transcriptomics revealed no systematic delay during early neural differentiation. This showed that differentiation can be uncoupled from growth and proliferation, suggesting alternative mechanisms. One candidate identified was the UDP-glucose pyrophosphorylase 2 (UGP2), required for glycogen synthesis. High UGP2 correlated with slow differentiation. Consequently, glycogen content was highest in human cells, intermediate in monkey and lowest in mouse. Thus, glycogen content is a species-specific cellular property that was unknown to this date. Neural differentiation of UGP2 knock-out cells revealed premature expression of the forebrain marker FOXG1, indicating that UGP2 could contribute to setting differentiation timing. It remains to be tested, how loss of UGP2 globally affects differentiation and by which mechanisms UGP2 acts. Taken together, these findings show that differentiation can be uncoupled from growth and cell cycling and implicate UGP2 and glycogen metabolism in the regulation of timing. This constitutes a novel mechanism by which cells could determine their differentiation speed.
Stereoselective synthesis of glycosides through novel catalytic method development
(2024) Wang, Caiming; Waldmann, Herbert; Strohmann, Carsten
Carbohydrates are one of the most prevalent natural product class with a wide range of structural and functional properties. Further, glycoside bond formation is one of the most important process in carbohydrate chemistry, particularly the production of O(S)-glycosides, which are abundant in bioactive compounds. To achieve this, glycosyl donors are synthesized and converted into reactive glycosylating species using catalysis. In this thesis, various activation strategies have been established to activate different glycosyl donors to synthesize a range of O(S)-glycosides, some of which are bioactive compounds. A phosphonochalcogenide (PCH) catalyzed strategy was developed to catalyze a stereoselective α-iminoglycosylation of iminoglycals with a wide range of glycosyl acceptors with remarkable protecting group tolerance in chapter 3. Mechanistic research revealed the catalyst's unexpected role in serially activating both the glycosyl donor and acceptor in the upstream and downstream stages of the reaction via chalcogen bonding (ChB). The dynamic interaction of chalcogens with substrates brings up new mechanistic possibilities based on repetitive ChB catalyst engagements and disengagements in multiple elementary steps. This research addressed the overall shortage of robust catalytic iminoglycosylations and provided a feasible approach for biologically relevant sp2-iminoglycosidic scaffolds. This methodology will demonstrate the enormously underexploited potential of sigma hole-based activation in broadening the frontiers of stereoselective carbohydrate and glycomimetic synthesis in the future. A synergistic chiral Rh(I) and organoboron-catalyzed protocol was introduced to catalyze site selective carbohydrate functionalization to synthesize biologically relevant anomeric aryl naphthalene glycosides with excellent enantioselectivity, diastereoselectivity and regioselectivity in chapter 4. The proper choice of an organoboron catalyst and ligands are critical to the success of this protocol. Following further investigation of this approach, my study revealed that structurally related allylic carbonate substrates were also well tolerated to furnish functionalized carbohydratesl with outstanding regio- and diastereoselectivity. This successful methodology would stimulate more effort in the development of chiral transition catalytic systems for demanding site-selective functionalizations of carbohydrates with prochiral electrophiles.
Mechanisms of spatial and temporal regulation of actin dynamics
(2024) Roy, Ankit Animesh; Bastiaens, Philippe I. H.; Kierfeld, Jan
Actin subunits in filaments exhibit accelerated ATP hydrolysis compared to monomers. The subsequent slow phosphate (Pi) release results in an age-dependent distribution of ADP-Pi and ADP subunits within the filament. This stochastic process prevents newer regions from severing, as turnover occurs primarily in older sections rich in ADP subunits. However, the mechanism underlying Pi release from actin filament cores remains elusive. In the initial portion of this study, we present the mechanism responsible for Pi egress from actin filament cores. Our findings indicate that Pi can readily escape through a molecular backdoor in the terminal subunit which is largely occluded in the core of the filament. Utilizing molecular dynamics simulations, we identified potential egress pathways for the exit of Pi from filament cores. These escape pathways appeared to be obstructed by an intricate network of hydrogen bonding involving residues previously associated with disease-linked mutations. Lastly, our study demonstrates that disrupting this hydrogen-bonding network with actin mutants that confer an open-backdoor conformation results in increased Pi release in both bulk polymerization and single filament measurements. The Arp2/3 complex initiates lamellipodial protrusions and endocytic actin patches through nucleation of force-generating branched actin networks. The activity of the Arp2/3 complex is regulated by Nucleation Promoting Factors (NPFs), characterized by a conserved C-terminal domain that activates the Arp2/3 complex and diverse N-terminal domains that exert spatial and temporal control over the NPFs. While some NPFs are autoinhibited and require the release of intramolecular autoinhibition by GTPases and phosphatidylinositol lipids for activation, others exhibit unclear regulatory mechanisms. Importantly, all NPFs assemble branched actin networks exclusively from cellular membranes. In the second section of this study, we propose a novel density-dependent mechanism for the regulation of NPF activity. We demonstrate that NPFs recruited to the membrane can spawn a branched actin network from a pool of profilin-actin under physiological concentrations of Arp2/3 complex and capping protein, whereas NPFs in solution cannot. Moreover, the nucleation process shows a switch-like response to membrane-bound NPF density, with nucleation only occurring above a threshold density. The concentration of capping protein can further tune this density-dependent response of NPF activity. Our findings suggest that this density-dependent regulation operates on NPFs regardless of autoinhibition, thus superseding the traditional autoinhibition mechanism.
Optogenetische Transkriptionskontrolle
(2024) Seifert, Swantje; Brakmann, Susanne; Summerer, Daniel
Licht als externer Stimulus zur Regulation der Proteinaktivität hat gegenüber klassischen Inhibitoren und Aktivatoren verschiedene Vorteile, wie z.B. eine sehr hohe räumliche und zeitliche Auflösung. Der Einsatz von photosensitiven Proteinen zur Untersuchung des dynamischen Verhaltens von intrazellulären Reaktionen und Reaktionskaskaden ist daher von großem Interesse. Photoaktive Proteine lassen sich in der Regel in zwei Module unterteilen. Die Sensordomäne, die mit einem lichtempfindlichen Liganden wechselwirkt und die Effektordomäne, die die Funktion des Proteins und die Signalweiterleitung bestimmt. Die Light-Oxygen-Voltage (LOV)-Domäne wurde in der Vergangenheit häufig als Sensor eingesetzt und mit verschiedenen Proteinen fusioniert, um deren Aktivität durch Licht zu regulieren. Es ist von entscheidender Bedeutung, eine geeignete Strategie zu finden, um die Sensor- und Effektordomänen so zu kombinieren, dass die Aktivität und die lichtabhängige Reaktivität erhalten bleiben. Für die T7-RNA-Polymerase wurden daher verschiedene Strategien getestet, um mit Hilfe der AsLOV2-Domäne eine optogenetische Kontrolle der Transkription zu erreichen. Es konnte gezeigt werden, dass die native terminale Fusion nicht universell anwendbar ist. Im Gegensatz zu dieser Strategie ist die Generierung lichtsensitiver T7-RNA-Polymerase-Varianten durch Insertion der Sensordomäne AsLOV2 möglich.
Tissue-intrinsic control of AVE differentiation in stem cell models
(2024) Schumacher, Sina; Bastiaens, Philippe; Trappmann, Britta
Differentiation and patterning in the early mammalian embryo depend on interactions between embryonic and extraembryonic lineages. One critical cell population specified through these interactions is the anterior visceral endoderm (AVE). Situated within the visceral endoderm (VE), the AVE serves as a pivotal signaling center during embryonic development by determining the body axis, ultimately positioning the head and tail within the underlying epiblast (Epi). The prevailing model for AVE differentiation in the mouse embryo posits that the AVE is induced by a uniform signal from the Epi, but an external inhibitory gradient from another extraembryonic tissue restricts its formation to a specific location beyond the gradient. In this thesis, I used mouse embryonic stem cells to establish an embryo-like aggregate system that challenged this model for AVE differentiation. These aggregates consist of an inner Epi core surrounded by a VE layer, thus resembling the embryo around the time of AVE formation but lacking the external gradient for AVE restriction. Remarkably, contrary to the established model, these aggregates manifest a spatially restricted AVE region within their VE compartment. Through single-cell RNA sequencing data analysis, I identified Nodal signaling as the critical signal from the Epi inducing AVE formation in the outer VE. Building upon this discovery, I initiated AVE differentiation in a homogeneous culture of VE cells, resulting in the development of a 2D AVE model. Activation of Nodal signaling within this model led to the formation of spatially constrained AVE clusters, unveiling a novel intrinsic mechanism within VE cells for spatially regulating AVE differentiation. To delineate the signal antagonizing AVE differentiation, I explored the role of different signaling networks in this tissue-intrinsic mechanism and identified β-catenin as the counteracting signal for AVE differentiation. Together, the findings shed light on an unexplored aspect of self-organization in AVE differentiation, potentially complementing the existing model of an inhibitory gradient mechanism in AVE formation.
Identification of transcriptional modulators of noise and differentiation in embryonic stem cells
(2024) Fernkorn, Max; Bastiaens, Philippe; Hengstler, Jan
During embryonic development, many cell types arise from a single homogenous cell population. Coordination via cell-cell signaling leads to functional specialization by changing the set of expressed genes. In very early mammalian development, the homogenous cell population of pluripotent cells in the inner cell mass breaks symmetry and differentiates into epiblast and primitive endoderm lineages under the influence of Fibroblast Growth Factor (FGF), Wingless Integrated (Wnt), and mammalian Target Of Rapamycin (mTOR) signaling. These signals modulate the activity of sequence-specific and general transcription factors. The involved sequence-specific transcription factors have not been identified for all mentioned signals and it is unclear whether there are specific components of the general transcriptional machinery which are especially important during early differentiation. Moreover, it is an open question how signals and general transcription factors cooperate to shape the transcriptional dynamics. Here, I ask how developmental signals are translated into transcriptional responses in pluripotent mouse embryonic stem cells (mESCs). I show that FGF4 signaling affects the transcriptional dynamics at target genes, resulting in an increase in cellular noise. I perform a genome-wide CRISPR screen to identify further signaling components and general transcriptional regulators for gene expression changes upon differentiation signals. Specifically, I focus on gene perturbations affecting the expression of a Spry4H2B-Venus reporter, which is upregulated in response to FGF signaling during the transition from naïve pluripotency to both epiblast and primitive endoderm cells. This screen returns multiple hits related to FGF signaling as expected and reveals general transcription factors associated with the Elongator and Mediator complexes. Focusing on Med12, a Mediator subunit whose loss affects the expression of the Spry4 reporter strongest, I find that it regulates gene expression during pluripotency transitions acting in parallel to FGF, Wnt, and mTOR signaling. During the exit of pluripotency, Med12-mutant cells react less efficiently to changes in the signaling environment both functionally and on the gene expression level. Surprisingly, the generation of epiblast and primitive endoderm cells is largely buffered against the loss of Med12. During this bifurcation, Med12-mutant cells show lower plasticity and noise levels, causing a better separation between the two cell types. These findings suggest that Med12 is an important general transcription factor during differentiation to prime transcriptional changes. Med12 acts in parallel to FGF-signaling in order to regulate the transcriptional variability and thereby allows cells to explore differentiation trajectories while keeping their plasticity.
Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles
(2023-12-15) Nanda, Suchet; Calderon, Abram; Sachan, Arya; Duong, Thanh-Thuy; Koch, Johannes; Xin, Xiaoyi; Solouk-Stahlberg, Djamschid; Wu, Yao-Wen; Nalbant, Perihan; Dehmelt, Leif
Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.
CRISPR-based screening & functional characterization of long non-coding RNAs in melanoma
(2024) Petroulia, Stavroula; Waldmann, Herbert; Watzl, Carsten
Melanoma, the most lethal form of skin cancer, is increasingly prevalent in Western populations and is characterized by its high metastatic potential. This cancer type is distinguished by a significant accumulation of somatic mutations, primarily due to UV radiation, leading to a mutation rate that exceeds that of most other solid tumors. The progression of melanoma involves the uncontrolled proliferation and spread of malignant melanocytes, with notable disruptions in the MAPK-ERK and PI3K-AKT-mTOR signalling pathways contributing to the development of advanced therapeutic strategies. Recent research has highlighted the importance of the non-coding regions of the genome, previously considered "junk DNA", for their regulatory functions. Long non-coding RNAs (lncRNAs), which are typically longer than 200 nucleotides, have been identified as key players in cellular development, differentiation, and cancer progression. Numerous studies have established a link between lncRNAs and the growth and progression of melanoma, with elevated lncRNA expression levels observed in melanoma cases. Herein, the present study attempted to elucidate the involvement of a specific set of lncRNAs in mechanisms underlying cell growth and proliferation. These lncRNAs, which had been observed to exhibit increased expression in melanoma cell lines and in short-term cultures derived from brain and lymph node metastasis, are examined by employing CRISPRi screening. Furthermore, functional characterization analyses were conducted on the most promising lncRNA candidates, including BDNF-AS, GMDS-AS1, and a novel, non-annotated lncRNA named XLOC030781 based on preliminary data that validate their importance in melanoma. Suppression of these lncRNAs led to apoptosis and inhibited cell cycle progression, with XLOC030781 playing a notable role in melanoma migration, broadening its functional scope in relation to this malignancy. Finally, the implementation of fluorescence in situ hybridization technique provided data on their subcellular localization, offering complementary information on their functionality.
Targeting RNA-protein interactions with peptides and small molecule inhibitors
(2023) Chang, Jen-Yao; Waldmann, Herbert; Dehmelt, Leif
WD repeat domain 5 (WDR5) is a scaffold protein involved in protein-protein or RNA-protein complexes, and most of these complexes play an important role in various epigenetic modulation processes. In particular, some of the long non-coding RNAs (lncRNAs) rely on the formation of the lncRNA-WDR5 complex to exert their epigenetic modulation, such as the upregulation of the lncRNA itself. If an oncogenic lncRNA relies on this pathway to maintain its expression level, it is possible to inhibit its positive feedback loop and thus reduce the expression of the oncogenic lncRNA. Therefore, this thesis focuses on investigating the potential of targeting lncRNA-WDR5 interactions, followed by evaluating the downregulation of lncRNA in cellulo. Targeting lncRNA-WDR5 interactions could be achieved by designing an inhibitor that targets the same binding pocket on WDR5. Several lncRNA are reported to recognize WDR5-binding motif (WBM) site, as a result, a study of structure-activity relationship of peptide-based inhibitors derivatize from protein-WDR5 interactions to target WBM site are performed. Further optimizations are performed by tailor-design macrocycle structure to facilitate peptides adopt the binding conformation and indeed the binding affinity is improved. The ability of macrocycle to disrupt lncRNA-protein interaction is verified by competitive in vitro RNA immunoprecipitation (iv-RIP). Cellular experiments are performed to determine whether targeting lncRNA-WDR5 interactions leads to downregulation of the lncRNA itself. Several strategies show their ability to enhance cell uptake of macrocycles. Finally, two molecules show that targeting lncRNA-WDR5 complexes can lead to reduction of the lncRNA itself and that different lncRNA have different sensitivity to the treatment.
A bacterial surface display platform for the discovery of cytosine modification readers from cDNA libraries
(2023) Schiller, Damian; Summerer, Daniel; Mutschler, Hannes
5-methylcytosine (5mC) occurs in palindromic cytosine guanine dyads (CpGs) of mammalian genomes and is a key element of epigenetic transcription regulation. Central to these regulatory functions is the ability of cytosine modifications to modulate the interaction of chromatin proteins with DNA. In this context, reader proteins are known to selectively recognize the cytosine modification. Interactome profiling studies based on pulldowns of nuclear extracts in combination with mass spectrometry-based (MS) proteomics have been the main approach to discovering readers of 5mC. However, this approach may miss important (anti-)readers due to the competition of proteins for the probe and low expression levels. Moreover, direct readers cannot be distinguished from indirect binders of protein complexes. In the scope of this thesis, a bacterial surface display system for discovering novel reader candidates from human cDNA was established. In detail, our approach offers screenings of cDNA-encoded protein libraries independent of their endogenous expression levels and without competition with other proteins. It further allows for rapid iterative FACS selections with defined, fluorescently labeled on- and off-target DNA probes. Furthermore, the system benefits from the fast protein expression machinery and growth of bacterial cells. We created surface display libraries of full-length and fragmented coding sequences (CDS) from human cDNA and demonstrated the compatibility of the Intimin surface display platform with displaying human proteins and protein fragments. Moving on, we proved the functionality of our selection system by enriching known 5mC reader candidates from a display library of human thyroid cDNA. In addition, we were able to identify novel reader candidates. Therefore, this study offers a promising tool to complement existing efforts in discovering 5mC reader candidates. Furthermore, the tool bears the potential to be extended to the oxidized derivates of 5mC and their combinations in CpGs, which have not been investigated so far.
Design, synthesis and biological evaluation of tool compounds for the cellular investigation of deubiquitinases
(2023) Schmidt, Mirko; Gersch, Malte; Waldmann, Herbert
The ubiquitin proteasomal system, is an important mediator of protein homeostasis through ubiquitination of substrates, but also coordinates various other functions throughout the cell. Deubiquitinases (DUBs) are essential regulators within this system. They are a specialized class of proteases that can cleave ubiquitin from its substrates and their dysregulation is involved in the development of various diseases. However, the understanding regarding their substrates, cellular localization, their involvement in cellular signaling and structural features remains limited. Despite the enormous therapeutical potential only a few DUB inhibitors are known to date. Therefore, novel tool compounds are of urgent need to enhance the understanding of DUBs in a cellular setting. This thesis describes the discovery, chemical synthesis and biological evaluation of such tool compounds for investigating DUBs in a living environment. To accomplish this, literature-known compounds were resynthesized to introduce alkyne tags to utilize their use as activity-based probes (ABPs) in cell-based systems. An intensive cellular characterization of a dedicated small molecule ABP library targeting DUBs resulted in the discovery of the chemogenomic pair of probes GK13S and GK16S, usable for the selective investigation of the DUB UCHL1 in a living environment. The probes were used to unravel potential substrates and a cellular phenotype after the inhibition of UCHL1. Furthermore, this thesis describes the synthesis and cellular evaluation of PROTAC molecules to study DUBs beyond inhibition. Altogether, this thesis should foster the understanding of DUBs and serve as a blueprint for the synthesis of further probe molecules to study these enzymes.
A cell fusion interaction network during the mating of Saccharomyces cerevisiae
(2023) Hagemeier, Angela; Raunser, Stefan; Westermann, Stefan
Cell-cell fusion is essential for sexual reproduction and occurs when the lipid membranes of two distinct cells merge into one continuous bilayer. While in recent years some general aspects have been uncovered, the underlying molecular mechanism remains poorly understood. Mating of haploid Saccharomyces cerevisiae cells of the opposite sex provides an ideal model system to study plasma membrane (PM) fusion in eukaryotic organisms. In this work, a multicolor flow cytometry assay based on fluorescent complementation (BiFC) of split-GFP was adapted to screen a customized yeast knockout library (YKO) for fusion defects. In total, 28 mutants were identified that exhibited fusion levels at least as defective as .prm1, a known regulator of this step. Like .prm1, the majority displayed a bilateral fusion defect. The remaining part of the work focused on an in-depth analysis of select gene of interest (GOI) mutants. Investigations of synergistic relationships in trans revealed an interaction network operating during PM fusion involving at least four independent yet partially overlapping fusion pathways. Previously two pathways with ERG6 and PRM1 have been reported. VMA2, a gene encoding a subunit of the vacuolar membrane ATPase (V-ATPase), was revealed in this work to operate on a third pathway. The findings show that the V-ATPase i) promotes cell fusion indirectly by acidifying endomembrane organelles, ii) facilitates both cell wall (CW) remodeling and PM fusion stages approximately equally, and iii) synergistically interacts with 12 other genes identified in this study. CAX4 was identified to operate on the fourth pathway and the only novel gene found to synergize with PRM1. Further investigations revealed that Prm1p was less abundant in a .cax4 sensitized background, while its localization is not affected. Finally, this work discovered that several subunits of the RNA polymerase II mediator complex are involved in promoting early and late stages of yeast mating. The deletion of subunits leads to varying degrees of defects in cell pairing and pheromone secretion as well as CW remodeling and PM fusion. Together, these findings suggest that the mediator complex acts as a master regulator of cell fusion perhaps by synchronizing the expression of mating genes needed at crucial time points starting from the digestion of the CW up to the merging of the PMs.

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