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TU Darmstadt | Plant biotechnology & metabolic engineeringThe core of our research is to understand the metabolic capacity of plants to build complex chemical structures, so-called specialized metabolites. With that knowledge we aim to engineer plants to produce a new set of metabolites, especially those which can serve as pharmaceuticals.
Background
Picture: Heribert Warzecha
Whether suffering an attack, luring pollinators, or basking in symbiotic comfort, plants constantly participate in molecular interplay with their environment. Stealthily sequestering dangerous poisons, freely exuding volatile attractants, or tirelessly sending and responding to various cues, they are versatile chemists producing myriad specialized compounds. Secondary metabolites thus come into view as the means of inter-organismal and intra-environmental communication in this dynamic continuum.
The biosynthetic capacity of plants is founded upon an intricate matrix of metabolic trails and rooted in a near inexhaustible supply of photosynthetic energy. It comes as little surprise then, that plants and plant-derived preparations have been used as commodities and remedies since time immemorial, and that the application of plant natural products (PNPs) in the development of new drugs and drug leads is still alive and well today.
Modulation of plant secondary metabolism: metabolic engineering
Picture: Heribert Warzecha
For any attempt at targeted engineering of biosynthetic routes leading to specialized compounds of therapeutic and/or economic value, it is of utmost importance to understand the biogenesis of PNPs from ubiquitous primary metabolites. Along with tremendous success in elucidation of several plant metabolic trails, their re-establishment in heterologous hosts has been a hallmark of recent endeavors in the field. However, current metabolic engineering efforts are, in the main, aimed at grafting the pathways to fermentable recipient organisms, like bacteria or yeast. Conducive to activity requirements of many catalytic proteins involved in the build-up of the complex carbon skeletons of interest, especially the latter has proved malleable enough to accommodate plant secondary metabolite production. Still, mere integration of biosynthetic genes into the eukaryote is not a sufficient driver for effective heterologous generation of PNPs in yeast as the issue of precursor supply necessitates substantive pathway engineering and, more often than not, retrieval rates of target compounds remain low, especially relative to their plant-produced counterparts. Conversely, while harboring orthologous metabolic routes, select plant species now emerge as viable vehicles for mobilization and engineering of complex biosynthetic pathways and manufacture of considerable amounts of high-value PNPs.
Drawing on the unique compartmentation of plant cells and capitalizing on the versatility of heterologous plant systems, our research team strives to re-wire them to generate new functionalities within the impressive repertoire of specialized metabolites. In concert with fundamental research aimed at ultimate deciphering of native biosynthetic pathways, we explore novel, combinatorial approaches affording production of new-to-nature, bespoke chemicals of potential commercial value.
Our investigative and engineering efforts focus on the biosynthetic networks yielding monoterpenoid indole alkaloids characteristic of the Indian snakeroot (Rauvolfia serpentina), phenolic secoiridoids indigenous to olive (Olea europaea), and cannabinoids, products of the secondary metabolism of Cannabis sp.
Susphire
We are involved in a new interdisciplinary project dedicated to sustainable bioproduction of pheromones for insect pest control in agriculture.
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Not just small molecules: chloroplasts as hubs of therapeutic protein production
Plastids, as relicts of endosymbiotic cyanobacteria, feature assorted prokaryotic traits. Chief among them are the efficiently functioning homologous recombination system and polycistronic organization of operons. Further, plant cells harbor a multitude of plastids, chiefly chloroplasts; these, in turn, carry multiple genome reprints. The enumerated characteristics thus set the stage for targeted and highly effective plastid genome manipulation.
In our efforts to harness their tremendous engineering potential, we view plastids as a coordinated system of mini-bioreactors within the larger context of a plant – a biofactory of therapeutically relevant recombinant peptides and proteins. TU Darmstadt | Synthetic RNA biologyWe explore regulatory RNAs in all of their diverse forms and shapes. Our research interests include natural RNAs found in bacteria and other model organisms as well as the design of aptamers and synthetic riboswitches. Our goal is to fully understand these regulatory elements in their structure, function and range of applications.
RNA performs many diverse and essential roles that considerably transcend the mere transmission of genetic information. The last decade saw the identification of a plethora of regulatory RNAs participating in crucial steps of gene expression. The molecular basis originates from the conformational flexibility and functional versatility of this macromolecule: like proteins, RNA can adopt complex three-dimensional structures for the precise presentation of chemical moieties that is essential for its function as a biological catalyst, regulator or structural scaffold.
Our key interest is to study the ways in which RNA exerts regulation. We search for unknown regulatory RNAs across different domains of life, create artificial ones and modify them for an in-depth understanding of their function and capabilities. Our goal is to comprehensively understand regulatory RNAs in all of their forms and use their potential in synthetic biology, medicine and biotechnology.
Aptamers, synthetic riboswitches and biosensors
Picture: Leon Kraus
A main focus of our research is the development of engineered riboswitches that can be applied as genetic regulatory devices for synthetic biology. These regulatory elements result from direct RNA-ligand interactions and are perfect model systems to study the molecular basis underlying this novel type of regulation. Riboswitches consist solely of RNA. They are characterised by binding of a small molecule effector (ligand?) to the so-called aptamer domain, which results in a conformational change of the downstream expression platform that determines output. The modular organisation of riboswitches and the possibility that small molecule-binding aptamers can be selected in vitro against almost any molecule of choice have recently led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology.
Picture: Adrien Boussebayle, Florian Groher
While many RNA aptamers binding to a multitude of small molecules have been identified, only very few are capable of acting as riboswitches. We therefore established a pipeline that integrates the in vitro selection process, next generation sequencing and in vivo screening for rapid identification of those aptamers that have the potential to be engineered into riboswitches. Thus, we implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. In a combination of genetic, biochemical and structural studies we address the question how regulation of riboswitching aptamers works at the molecular level. Finally, we explore applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression and to develop new strategies e.g. for controlling RNA stability or alternative splicing by synthetic riboswitches in bacteria, archaea and eukaryotes.
In addition, we also employ synthetic riboswitches to build logical gates and complex genetic circuits. For these purposes, we like to use bioinformatic support such as machine or deep learning. Due to their excellent binding properties, aptamers are also well suited as biosensors, for example to detect contamination with pollutants, antibiotics or drug residues in food or drinking water. Here we develop easy-to-use, fast and safe biosensors.
Finding regulatory RNAs in Streptomyces
Streptomycetes are highly relevant soil bacteria in biotechnology best known for their complex life cycle and ability to produce a wide range of secondary metabolites. Using RNAseq, we identified and characterized several small non coding RNAs (sRNAs) in the model organism Streptomyces coelicolor. Our specific focus lies on sRNAs that potentially affect the production of secondary metabolites, such as antibiotics and other pharmacologically active compounds. TU Darmstadt | Protein engineering: signalling and transportOur research group takes a protein-centric approach to synthetic biology as we devise systematic approaches to engineer artificial sensory and transport functions. Strategically, we address both fundamental research questions and develop applications for industrial biotechnology and biomedicine. We tackle these questions through a combination of molecular-genetic, biochemical and biophysical methods.
General Strategy
To engineer artificial sensory and transport functions, our group specifically aims to (i) develop foundational technologies for protein engineering which includes new DNA assembly procedures and genetic screening systems, (ii) dissect the sequence-structure-function relationships underlying artificially engineered protein functions, (iii) map the evolutionary trajectories that connect distinct protein functions, and finally (iv) develop molecular tools for different applications in biotechnology, biomedicine and basic research.
Engineering synthetic protein switches, sensors and signalling circuits
Our primary goal concerns the construction of synthetic protein switches, sensors and thereof derived signalling circuits with tailored response functions. A specific area of interest concerns the development and application of tailored fluorescent protein sensors and protease switches. In particular, our group focusses on the role of linkers that underlie conformational transitions and other key functional properties of artificially engineered protein switches and sensors such as expression and folding. To facilitate linker engineering, we recently developed a linker toolbox along with a new DNA assembly process – termed iterative functional linker cloning (iFLinkC) – tailored to the needs of proteins. Combinatorial library screens have since demonstrated a large plasticity underlying functional linker space and highlighted the potential of unconventional Pro-rich linkers that frequently yield the most potent switches and sensors. Additional efforts now focus on implementing tailored sensors, switches and circuits in live cells for different biotechnological application. This is complemented by foundational projects that aim to examine how the operation of synthetic protein switches, sensors and circuits in live cells impacts host cell physiology.
Engineering transport across biological membranes
Our second goal concerns the construction of artificial transport functions across biological membranes. Here, progress has been hampered by a lack of (i) molecular toolboxes of well characterised molecular components to construct tailored transport function, and (ii) genetic screening systems that enable us to assay nanopores, ion channels and transporters in high-throughput. Addressing these limitations, we recently developed a Functional Nanopore (FuN) screen to assay the functional properties of nanopores, ion channels and transporters in high-throughput. The assay features an optical read-out and can be applied across a range of experimental formats including conventional microtitre plates, on-plate colony, FACS and microfluidics. Current efforts aim to dissect the molecular and genetic factors that underlie the assembly, stoichiometry, permeability, specificity and size of pore-forming membrane peptides. Building on these foundational protein engineering efforts, our mid-term goal is to develop molecular toolboxes for nanopore engineering and develop them for different biotechnological applications.
Engineering transport across nanoporous membranes
Our third goal aims to develop systematic approaches to functionalise track-etched nanopores with tailored sensors, switches and receptors. Compared to biological membranes, nanoporous membranes are substantially more robust, but capable of generating equally sensitive and specific electrical read-outs with many potential applications as medical biosensors, in particular point-of-care diagnostics. Towards this goal, we recently developed a general approach to functionalise track-etched, nanoporous polymer foils with antibody fragments achieving quantitative and ultrasensitive electrical responses for a variety of analytes. Building on this capacity, we now aim to functionalise nanoporous materials with recombinant proteins in a systematic fashion. This includes both foundational studies and the developing biomedical applications looking to exploit the exquisite diversity of natural protein functions and examine how functionalisation impacts the responsiveness of track-etched nanopores in the context of biomedically-relevant analytes. TU Darmstadt | Microbial energy conversion & biotechnologyOur laboratory studies microbially catalysed reactions of the biogeochemical nitrogen and sulfur cycles at the cellular and subcellular levels. We are mainly interested in the physiology and bioenergetics of anaerobic/microaerobic bacteria and focus on the enzymology of anaerobic respiration and the functional architecture of corresponding electron transport chains.
Respiratory reduction of laughing gas
Picture: Jörg Simon
Model of the electron transport chain from menaquinol to laughing gas in a laughing gas-respiring bacterium containing a clade II nos gene cluster encoding cytochrome c nitrous oxide reductase (cNosZ), for example W. succinogenes. More information: Hein and Simon (2019) Bacterial nitrous oxide respiration: electron transport chains and copper transfer reactions. Adv. Microb. Physiol. 75, 137-175.
An environmentally important molecule of the biogeochemical nitrogen cycle is nitrous oxide (N2O, laughing gas), which is a potent greenhouse gas and an ozone depleting substance. We study the N2O metabolism of nitrate/nitrite-ammonifying and denitrifying bacteria that are capable to grow by nitrous oxide respiration. A prime example for this type of energy metabolism is the non-pathogenic rumen bacterium Wolinella succinogenes. As this bacterium is known to generate small amounts of nitric oxide (NO) and nitrous oxide during nitrate ammonification, we also explore the enzymology of nitric oxide detoxification and nitrosative stress defence.
The terminal reductase of W. succinogenes nitrous oxide respiration is a cytochrome c nitrous oxide reductase (cNosZ) that belongs to the so-called clade II of NosZ enzymes. We presented evidence that functional N2O respiration involves a menaquinol-oxidizing Rieske/cytochrome bc complex and various Nos proteins encoded in the nos gene cluster. Furthermore, transcriptome analysis suggested that N2O-grown W. succinogenes cells specifically regulate gene expression in response to nitrous oxide.
Over the last century the atmospheric concentration of nitrous oxide has been constantly on the rise. This fact is caused by the invention of man-made nitrogen fixation (the Haber-Bosch process), which led to a drastic intensification of agricultural fertilization practices and a severe anthropogenic imbalance of the global biogeochemical nitrogen cycle. In a biotechnological approach we aim to apply suitable N2O-respiring bacteria to different natural habitats to reduce N2O emissions.
We use a wide range of methods in cellular and molecular microbiology in combination with genetic engineering of suitable model bacteria, omics techniques, protein biochemistry and structural biology.
MccA-dependent sulphite respiration
Picture: Oliver Einsle
Structural model of the homotrimeric cytochrome c sulfite reductase MccA and its 24 bound haem c groups (monomers shown in blue, green and red; haem c groups in yellow). More information: Hermann et al. (2015) Octaheme MccA is a heme c:copper sulfite reductase. Nature 520, 706-709.
Sulphite is reduced to sulfide by the highly active octahaem cytochrome c sulphite reductase MccA, present in the periplasm of certain Proteobacteria. The high-resolution crystal structure of Wolinella succinogenes MccA revealed an unprecedented haem c-copper active site of sulfite reduction and confirmed the presence of a haem c group bound to a unique CX15CH haem attachment site.
Currently, we explore the constituents of the corresponding electron transport chain and the process of MccA maturation, which is supposed to include the putative copper chaperone MccL.
Biosynthesis and function of methylated menaquinone derivatives
Picture: Jörg Simon
Structure of 7,8-dimethylmenaquinone (7,8-DMMK). More information: Hein et al. (2018) Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone Biochim. Biophys. Acta 1859, 300-308.
The membranous quinone/quinol pool is an essential component of most bacterial and archaeal respiratory electron transport chains. Beside the well-known ubiquinone and menaquinone (MK), many bacteria produce mono- and dimethylated MK derivatives such as 8-methyl-MK and 7,8-dimethyl-MK.
We identified and characterized a family of HemN-related class C radical S-adenosylmethionine methyltransferases designated MenK, MenK2 or MqnK that methylate MK specifically at position C-8 (MenK/MqnK) or C-7 (MenK2). The MenK and MenK2 enzymes from Adlercreutzia equolifaciens were functionally produced in Wolinella succinogenes or Escherichia coli cells. We currently explore the reaction mechanism of these enzymes and the cellular function of methylated menaquinones.
TU Darmstadt | Biology of eukaryotic gene and genome regulationHow can we precisely perturb and control molecular processes in living eukaryotic cells? Our lab approaches this question from a synthetic biology and protein engineering perspective. We juxtapose various methods, namely optogenetics, CRISPR, viral vectors and computation and develop molecular tools to study genome regulation and direct cell function.
Picture: Dominik Niopek
Our lab has three research focus areas that are highly complementary and closely intertwined. First, we develop optogenetic tools based on photosensory proteins from nature. We invented a unique technology based on engineered, light-switchable anti-CRISPR proteins that allows controlling CRISPR-Cas genome editing and gene targeting. This technology facilitates spatiotemporally precise (epi)genome perturbations in mammalian cells, which we employ to study human genome regulation and function. Second, we create cell type-specific Adeno-associated virus (AAV) vectors for the efficient and safe delivery of CRISPR components into patients suffering from genetic disorders. Third, we combine artificial intelligence and high-throughput design to accelerate and eventually rationalize the engineering of proteins with novel properties.
Engineering anti-CRISPR proteins for conditional activation of CRISPR-Cas
Picture: Dominik Niopek
Anti-CRISPR proteins are potent inhibitors of CRISPR effectors. We engineer light-switchable derivatives of natural anti-CRISPRs by embedding photosensor domains, such as the light-oxygen-voltage 2 (LOV2) domain from Avena sativa, into Acr structures (Bubeck & Hoffmann et al., Nat. Methods, 2018; Hoffmann and Mathony, bioRxiv, 2019). The resulting, chimeric inhibitors block Cas9 in the dark, but release its activity upon blue light irradiation.
This approach which we named CASANOVA, for CRISPR-Cas activity switching by a novel, optogenetic variant of anti-CRISPR proteins, facilitates light-dependent genome editing and epigenome editing and is used in our lab to study genome regulatory processes. While CASANOVA was initially limited to the S. pyogenes Cas9, we are currently extending this original strategy to other Cas9 orthologues, Cas12 and Cas13, the latter of which is an RNA-targeting CRISPR effector. We are also developing designer Acrs that outperform their natural counterparts with respect to inhibition potency (Mathony, Harteveld and Schmelas et al., Nat. Chem. Biol., 2019).
Recently, we started to extend our protein engineering efforts towards a variety of other protein classes of interest for basic research and biotechnology. To render these proteins switchable upon light or chemical induction, we combine computational approaches with mutational scanning, a method for saturating mutagenesis and high-throughput functional analysis of protein candidates.
Developing safe and efficient AAV-CRISPR vectors
Picture: Dominik Niopek
One of our central aims is to support the clinical translation of the CRISPR technologies by developing safe and efficient AAV-CRISPR vectors (Senis et al., Biotechnology J, 2014). Towards this end, we use synthetic biology approaches to create switches and circuits that confine Cas9 activity to selected cell types, e.g. by coupling Cas9 activity to the abundance of cell type-specific microRNAs (Hoffmann et al., Nucleic Acids Research, 2019). We also combine wet lab experiments with mathematical modeling to fine-tune Cas9 activity to desired levels, thereby enabling the kinetic insulation of ON- and OFF-target editing events (Aschenbrenner & Kallenberger et al., Science Advances, 2020).
Machine learning-guided protein design
Picture: Dominik Niopek
By training neural networks on thousands of protein sequences and corresponding 3D protein structures, we aim at creating algorithms that can inform protein design (Upmeier zu Belzen et al., Nature Machine Intelligence, 2019). Applications range from the prediction of engineering hotspots to facilitate the development of switchable proteins to the re-design of enzymes and – in the future – the creation of immune “stealth” protein therapeutics. TU Darmstadt | Systems biology of the stress responseMammalian cells are constantly challenged with different forms of stress that originate from both the physical environment and intrinsic biological processes. We investigate how cells sense and counteract these stresses using a systems biology approach based on quantitative experiments in living cells, computer-aided data analyses and mathematical modelling.
Quantitative single cell biology
Individual cells within a population often react differently to the same stress, depending on their initial state. We therefore focus on the analysis of individual cells and investigate the common properties that unite them and the sources of variation that make them different. We use automated time-lapse microscopy of living cells expressing fluorescent reporters to measure the dynamics of signaling networks with high temporal resolution. Since signaling networks contain complex, non-linear interactions that are difficult to understand intuitively, we combine the resulting quantitative data with statistical analysis and mathematical modeling to gain a predictive understanding of cellular stress responses.
The p53 – mediated response to genotoxic stress
The tumor suppressor p53 is a central hub in the mammalian stress response, which coordinates the cellular defense by changing the expression of hundreds of genes that are involved in repair pathways, cell cycle arrest or the induction of senescence and apoptosis. The tumor suppressor itself is controlled by a complex network of positive and negative regulators. If this crucial signaling pathway is disturbed by mutation, cancer arises.
The main focus of our research is to understand the molecular mechanisms that enable p53 and its interacting pathways to faithfully manage the cellular stress response. We investigate how the underlying molecular networks act dynamically in living cells, how they control gene expression on a molecular level and how they intersect with each other to control the physiological response of a cell.
The dynamics of DNA double strand break repair in individual cells
Ionizing radiation leads to DNA double strand breaks that compromise the inherent fail-safe mechanism of the genome and threaten its integrity. To counteract these lesions, several sophisticated repair pathways evolved. We aim to quantify the dynamics of these repair mechanisms in individual living cells, explore the underlying molecular mechanisms and investigate their integration with other cell regulatory processes such as cell cycle control.
SMAD – mediated signaling in health and disease
Ligands of the TGFb superfamily regulate numerous cellular processes during adult homeostasis and regeneration by binding to transmembrane receptors and inducing nuclear translocation of signaling intermediates called SMADs. Alterations in SMAD – mediated signaling can cause diseases such as cancer and fibrosis or vascular malformations. We aim to understand how cells encode the information contained in these extracellular signals, how they use it to elicit cell-specific responses and how disease-associated mutations affect signal transduction in individual cells. We focus on the role of TGFb signaling in controlling proliferation and motility in human breast epithelial cells and the anti-angiogenic effect of BMP9 and 10 in human endothelial cells. TU Darmstadt | Radiation biology and DNA repairOur laboratory investigates the repair of DNA double-strand breaks (DSBs), the most toxic lesions endangering cellular viability. We are interested in dissecting DSB repair mechanisms and uncovering novel repair factors, providing basis to understand the pathogenesis of diseases such as cancer and the development of novel radio- and chemo-therapeutic strategies.
DSB repair around the clock
DSBs arise constantly in our cells by endogenous cellular mechanisms or are induced by exogenous agents, including ionising radiation (IR) and chemotherapeutics. Cells react to these lesions by inducing cell cycle arrest and initiating DSB repair mechanisms. The faithful repair of these breaks is critical for cell survival and genomic integrity. Incorrect repair leads to the accumulation of mutations and the development of diseases such as cancer. Furthermore, incomplete or inactive repair leads to the persistence of unrepaired breaks and subsequent induction of cell death, a process often exploited for cancer therapy. Therefore, elucidating DSB repair mechanisms is central to understanding the pathogenesis of diseases and evaluating and improving the efficacy of treatment. Since breaks are channelled into different repair mechanisms depending on the cell-cycle phase, we employ cell cycle-specific analysis of DSB repair to gain a more accurate knowledge of these processes. We use various cell and molecular biology techniques to study repair factors and their role in response to IR-, chemotherapeutics- and enzyme-induced DSBs in multiple cell systems.
Picture: Michael Ensminger
Targeting homologous recombination for cancer therapy
Homologous recombination (HR) is an error-free DSB repair pathway that is active in the S/G2 phase of the cell cycle and is the main pathway repairing spontaneously arising breaks. HR is often inactivated in cancer, a feature that has been central to various therapeutic strategies. Our lab has identified various novel factors involved in HR regulation, function and HR sub-pathway choice. We also characterized the usage of distinct HR sub-pathways in specific types of cancer compared to normal cells. Current projects involve the advancement of these findings to in vivo mouse models and to the pre-clinical evaluation of targeting these factors. We are particularly interested in identifying novel synthetic lethality relationships that could open up opportunities for therapeutic intervention.
Whole-genome mapping of NHEJ mechanisms and fidelity
Non-homologous end-joining (NHEJ) is a main DSB repair pathway operating throughout the cell cycle and is known to be error-prone. The compromised fidelity of this pathway is believed to cause mutations that likely drive cancer initiating events. Our lab identified a sub-pathway of NHEJ that shares features with HR and harbours potential for error-free repair. We are currently investigating factors affecting choice of NHEJ sub-pathways as well as the mechanisms driving enhanced repair accuracy. Current projects employ next-generation sequencing (NGS) for the genome-wide mapping of mutational signatures of NHEJ to identify mutation rates in wild-type and NHEJ-mutant cells. We also use chromatin immunoprecipitation sequencing (ChIP-Seq) to elucidate repair mechanisms operating at specific genomic loci and the influence of the chromatin landscape on repair pathway choice.
Evaluation of cellular responses following low-dose irradiation
While we encounter high doses of IR in specific situations, such as radiotherapy, we are constantly exposed to low IR doses in our daily lives from sources such as background radiation (e.g. radon) and routine medical examinations (e.g. X-ray diagnostics). Our work revealed that cells do not repair lesions from such exposure efficiently and possibly use distinct mechanisms from those handling higher levels of damage. This discrepancy could underlie an enhanced risk to low-dose IR exposure, in the form of increased mutations or, alternatively, can represent a protective mechanism where damaged cells are eliminated by cell death. Therefore, it is important to study the exact molecular processes involved in these responses in order to improve IR risk assessment and radiation protection procedures. TU Darmstadt | Neurophysiology and neurosensory systemsThe research in the Laube Lab focuses on the structure and function of biological sensors involved in synaptic transmission, neuronal development and cancer progression by combining biochemical, genetic, behavioral and electrophysiological methods. The knowledge about structural constraints of biological sensors will allow the construction of electrical BioSensors in biomimetic nanopores.
Synaptic transmission between nerve cells is mediated by structurally distinct families of ligand-gated ion channels which are differentially expressed in the brain and, remarkably, also in cells of diverse cancers. Thus, these channels are involved in both, i) brain function and malfunction, such as memory formation and neurodegeneration, and ii) in the stress response and in survival strategies of tumor cells, i.e. malignant progression or radio resistance. As these channels constitute targets of clinically relevant drugs, we intend to find perspectives for the development of therapeutically useful strategies for the treatment of neurological diseases and malignant brain tumors. In addition, based on the knowledge about the structural constraints determining the function and pharmacology of receptors, we use coupling to biological and artificial nanopores for the rational design of synthetic BioSensors employed for analyzing substances of biological interest.
Role of synaptic proteins in neurological diseases and developmental disorders
Neurotransmitter receptors represent key players in the physiology and pathophysiology of neurons during development and the maintenance of cognitive functions in the brain. Inappropriate activation induces developmental malformations, neuronal loss as well as certain neurodegenerative diseases. Mutations in these proteins are associated with a variety of different neurodevelopmental phenotypes, including intellectual disability (ID) and epilepsy. By analyzing structural and functional consequences upon heterologous expression of missense mutations in receptors associated with neurological diseases, we hope to get insights into the underlying pathological mechanisms.
Role of cellular stress responses and ionizing radiation in neuronal development
In the developing and adult brain, neural stem cells (NSCs) are characterized by the ability of self-renewal and to differentiate to neurons, astrocytes and oligodendrocytes. Evidences indicate that neurotransmitters and ionizing radiation influence neurogenesis in the hippocampus, a brain region involved in learning and memory processes, by promoting neuronal differentiation, death or survival by oxidative and nitrosative stress. We use mouse models to analyze the consequences of IR on behavior and different learning paradigms to characterize spatial learning abilities. Subsequent immunohistochemical evaluation of irradiated brains serves as a link between the systemic behavioral approach and molecular biological DNA damage analyses.
Role of membrane receptors in cellular stress response and tumor progression
We attempt to understand the function of receptors as part of the cellular stress response in cancer cells and how extracellular signaling interferes with brain tumor metastasis. Several tumors can secrete the excitatory neurotransmitter glutamate to stimulate their cell survival and to cause excitotoxic neuronal cell death in the surrounding tissue. Glutamate receptors expressed in these tumor cells promote tumor survival, metastasis, and endow tumors with an enhanced resistance to radiation- and chemotherapy. Thus, we examine the impact of glutamate and IR on cell survival and intracellular signaling pathways regulating gene expression and DNA damage response (DDR) especially in tumorigenic subpopulations, which are in focus of new therapeutic approaches.
Design and use of BioSensors
As ligand-gated ion channels in cell membranes represent biological nanopores converting physical, biological and chemical signals into robust current signals, we try to engineer new BioSensors on the basis of modified neurotransmitter receptors and binding proteins. The aim is to rational design specific BioSensor modules for stable integration into solid-state pores and subsequent incorporation in a lab on a chip system for medical and environmental analysis of substances of biological interest. 