Structural Biology (Hiller)Head of Research Unit Prof. Dr.Sebastian HillerOverviewMembersPublicationsProjects & CollaborationsProjects & Collaborations OverviewMembersPublicationsProjects & Collaborations Projects & Collaborations 22 foundShow per page10 10 20 50 Protein-protein interaction antagonists Research Project | 1 Project MembersNo Description available EMBO Fellowship for Patrick Fischer Research Project | 1 Project MembersEMBO Fellowship for Patrick Fischer Structure and mechanism of Leptospira's virulence switch Research Project | 1 Project MembersStructure and mechanism of Leptospira's virulence switch Mechanistic dynamics of the chaperone network in the endoplasmatic reticulum Research Project | 1 Project MembersMechanistic dynamics of the chaperone network in the endoplasmatic reticulum Structure-function analysis of the RNA-binding protein PGC-1a in skeletal muscle Research Project | 2 Project MembersPlasticity of cells, tissues and organs relies on sensing of external and internal cues, integration of the engaged signaling pathways, and orchestration of a pleiotropic response. For example, contractile patterns, ambient temperature and oxygen levels, nutrient availability and composition, as well as other factors result in a massive remodeling of biochemical pathways, cellular metabolism and mechanical properties of skeletal muscle cells, most notably in the context of repeated bouts of exercise, ultimately leading to training adaptations. In turn, such adaptations of skeletal muscle trigger internal and external changes that contribute to the systemic effects of exercise with many health benefits, and strong preventative and therapeutic outcomes in a number of pathologies. Surprisingly, even though the physiological and clinical contributions of exercise-linked muscle plasticity are well recognized, the molecular mechanisms that control the corresponding biological programs are still poorly understood. In recent years, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has emerged as a central regulatory nexus of plasticity in various cell types. PGC-1α integrates upstream signaling pathways to coordinate the complex transcriptional networks encoding biological programs involved in mitochondrial function, cellular metabolism and more. In the case of endurance exercise-mediated plasticity of muscle cells, most, if not all of the adaptations are evoked by this stimulus. Accordingly, muscle-specific overexpression or knockout of PGC-1α elicits high endurance and pathologically sedentary phenotypes, respectively. Our proposal aims at elucidating the molecular underpinnings of the complex transcriptional control that is exerted by PGC-1α in a spatio-temporal manner. We have recently discovered that the binding of RNAs to PGC-1α contributes to full transcriptional activity, and is central for the inclusion of PGC-1α-containing multiprotein complexes in liquid-liquid phase separated nuclear condensates for the sequestration of transcription. We now plan to determine the list and common features of PGC-1α-bound RNAs, interrogate how RNA binding is brought about in a structure-function analysis, study the consequences on multiprotein complex formation, and ultimately investigate the physiological relevance in skeletal muscle cells in vitro and in vivo . To do so, novel animal models will be leveraged for single-end enhanced crosslinking and immunoprecipitation (seCLIP) experiments to identify PGC-1α-bound RNAs, structural information will be obtained by NMR spectroscopy, and validated using site-directed mutagenesis of key amino acids and nucleotides in PGC-1α and RNAs, respectively, to study PGC-1α-dependent liquid-liquid phase separation. Then, PGC-1α-containing multiprotein complexes will be co-purified to perform cryo-electron microscopy-based single particle structural analysis. Finally, the functional consequence of targeted mutagenesis of key residues will be assessed in muscle cells in culture and mouse muscle in vivo with state-of-the-art myotropic adeno-associated viral vectors (AAVMYO). The research plan thus a.) highly synergizes in terms of interdisciplinary approaches between the groups involved and b.) will result in hitherto unprecedented insights into molecular mechanisms that underlie complex spatio-temporal control of transcriptional networks with important implications for the understanding of cellular and tissue plasticity in health and disease. NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria Umbrella Project | 32 Project MembersAntibiotics are powerful and indispensable drugs to treat life threatening bacterial infections such as sepsis or pneumonia. Antibiotics also play a central role in many other areas of modern medicine, in particular to protect patients with compromised immunity during cancer therapies, transplantations or surgical interventions. These achievements are now at risk, with the fraction of bacterial pathogens that are resistant to one or more antibiotics steadily increasing. In addition, development of novel antimicrobials lags behind, suffering from inherently high attrition rates in particular for drug candidates against the most problematic Gram-negative bacteria. Together, these factors increasingly limit the options clinicians have for treating bacterial infections. The overarching goal of NCCR AntiResist is to elucidate the physiological properties of bacterial pathogens in infected human patients in order to find new ways of combatting superbugs. Among the many societal, economic, and scientific factors that impact on the development of alternative strategies for antibiotic discovery, our limited understanding of the physiology and heterogeneity of bacterial pathogens in patients ranks highly. Bacteria growing in tissues of patients experience environments very different from standard laboratory conditions, resulting in radically different microbial physiology and population heterogeneity compared to conditions generally used for antibacterial discovery. There is currently no systematic strategy to overcome this fundamental problem. This has resulted in: (i) suboptimal screens that identify new antibiotics, which do not target the special properties of bacteria growing within the patient; (ii) an inability to properly evaluate the efficacy of non-conventional antibacterial strategies; (iii) missed opportunities for entirely new treatment strategies. This NCCR utilizes patient samples from ongoing clinical studies and establishes a unique multidisciplinary network of clinicians, biologists, engineers, chemists, computational scientists and drug developers that will overcome this problem. We are excited to merge these disciplines in order to determine the properties of pathogens infecting patients, establish conditions in the lab that reproduce these properties and utilize these in-vitro models for antimicrobial discovery and development. In addition, clinical-trial networks and the pharmaceutical industry have major footprints in antimicrobial R&D. Exploiting synergies between these players has great potential for making transformative progress in this critical field of human health. This NCCR maintains active collaborations with Biotech SMEs and large pharmaceutical companies with the goal to: accelerate antibiotic discovery by providing relevant read-outs for early prioritization of compounds; enable innovative screens for non-canonical strategies such as anti-virulence inhibitors and immunomodulators; identify new antibacterial strategies that effectively combat bacteria either by targeting refractory subpopulations or by synergizing with bacterial stresses imposed by the patients' own immune system. This NCCR proposes a paradigm shift in antibiotic discovery by investigating the physiology of bacterial pathogens in human patients. This knowledge will be used to develop assays for molecular analyses and drug screening under relevant conditions and to accelerate antibacterial discovery, improve treatment regimens, and uncover novel targets for eradicating pathogens. Through this concerted effort, this NCCR will make a crucial and unique contribution to winning the race against superbugs. Development of NLRP3-ASC selective inhibitors Research Project | 1 Project MembersDuring an attack from pathogens organisms produce inflammatory cytokines, such as IL-1β and IL-18, that act to combat infection. However, prolonged and uncontrolled production of inflammatory cytokines, usually due to sterile inflammatory triggers, causes chronic inflammation. The latter is the underlying cause of several (auto)-inflammatory diseases such as gout, CAPS (Cryopyrin-Associated Periodic Syndromes), arthritis, Alzheimer's disease, etc. The main cellular node activated by sterile (auto)-inflammatory triggers is NLRP3-inflammasome (NACHT, LRR and PYD domains-containing protein 3). Main drugs against chronic inflammation act downstream of the NLRP3-inflammasome by targeting the release of inflammatory cytokines, with variable efficacity. However, these drugs block the entire inflammatory response, as such, an increase in the rate of infections is a main concern. Alternatively, blocking specifically the NLRP3-inflammasome would inhibit only one branch of the innate immune response having an immense potential to treat (auto)-inflammatory disorders with fewer side effects. As of today, there are no NLRP3-inflamasome targeting drugs on the market. To develop such an inhibitor, we are focusing on 11 small molecules obtained from a cell-based screen of over 10'000 compounds. Our small molecules have novel chemistry to the known NLRP3 inhibitors and, without optimization, show NLRP3-inflammasome inhibitor activity comparable to known NLRP3 inhibitors. During the course of this feasibility study, we will perform hit-to-lead optimization and find the molecular mechanism of action of our compounds. We aim for a business-to-business model. By combining the expertise of our team in structural biology, immunology, organic chemistry and animal experimentation we will generate intellectual property that we can patent, and use as leverage to find business partners to push our compounds towards Phase I clinical trials. A versatile technology platform for identification and development of novel bio-antibiotics Research Project | 1 Project MembersThe rise of multidrug-resistant bacteria threatens the achievements of modern medicine. Particularly problematic in this context is the increasing resistance of the pathogens Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii and Pseudomonas aeruginosa . Antibiotics with novel modes of action are urgently needed to combat these bacterial species. We will establish a technology that enables the identification of high-affinity binders for targets on the surface of these pathogens. These binders are potential novel antibiotics. As a key application, we will target the outer membrane protein insertase BamA. Using the platform, we will select single-domain antibodies, which will be generated either by immunization of alpacas (nanobodies) or in vitro from synthetic nanobody libraries (sybodies), and we will then screen large libraries of such binder molecules for antibiotic activity. We refer to such biomolecular drugs against pathogenic bacteria as "bio-antibiotics". Our technology platform rests on two pillars. In a first step, sybody and nanobody selections are carried out against a purified sample of the target. Notably, only a small subset of the resulting binders will bind the OMP targets in the native context of the bacterial outer membrane, where some accessible epitopes are covered underneath a dense lipopolysaccharide layer. Therefore, in a second step our recently developed binder screening and characterization technology NestLink is applied, which is uniquely available to the Seeger lab. NestLink allows to deep-mine an enriched binder pool in a single experiment, which is impossible by conventional technologies. The utility of our bio-antibiotic technology platform will be demonstrated by the identification of lead molecules that inhibit the growth of clinical Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii and Pseudomonas aeruginosa strains. Suitable lead molecules will enter pre-clinical trials to treat life-threatening blood stream infections and frequently observed urinary tract infections. High-throughput functional assay for BAM-mediated protein folding in a native membrane Research Project | 1 Project MembersFor the development of novel antibiotics against Gram-negative bacteria, targets in the outer membrane or of prime interest. There exist two generally essential such targets, the outer membrane protein insertase BAM with the core unit BamA and the LPS insertion machinery LptD with its associated proteins. Two novel classes of inhibitors for BamA, a synthetic as well as a natural antibiotic, were recently discovered (Luther et al, Nature 2019, Imai et al, Nature 2019). This validity of BAM as a target creates an imperative requirement of an assay to screen for further inhibitors of BAM catalytic activity in vitro . While assays with synthetic vesicles have been reported, so far, no assay for studying BAM-catalyzed OMP folding in the native membrane is available. A quantitative assay to study OMP folding in the natural bacterial membrane has recently been developed by teh Hiller lab in Biozentrum, University of Basel. The assay makes use of outer membrane vesicles (OMVs) that are released from Gram-negative bacteria as spherical buds from the outer membrane, and hence contain the lipid composition and distribution of the outer membrane. They thus provide native and physiological conditions to study the OMP biogenesis mechanism. It is the goal of this collaborative project to establish the assay for high-throughput screening. The assay is expected to allow screening of novel antibiotics, as well as hit validation and lead expansion in high-throughput format. Biophysical Principles of Chaperone-Client Interactions Research Project | 1 Project MembersViele Proteine sind im zellulären Kontext auf die korrekte Entstehung ihrer räumlichen Struktur angewiesen. Diese Struktur können sie oft nur mit Hilfe von anderen Proteinen, sogenannten Chaperonen erreichen. Fehlfaltung und Aggregation bereiten dem Organismus grosse Probleme, die zum Versagen ganzer Organe führen können. Da die Proteinkomplexe aus Chaperonen und ihren Substraten hochdynamisch sind, ist es schwierig, quantitative Beschreibungen davon zu erhalten. Diese sind aber essentiell, um Chaperone genau zu verstehen. Mit unserem Projekt wollen wir zu einem besseren molekularen Verständnis dieser wichtigen Proteinklasse beitragen. Inhalt und Ziele Mit unserem Projekt verfolgen wir zwei Hauptziele. Zum einen möchten wir neue Methoden entwickeln, um die Komplexe aus Chaperonen und Ihren Substraten quantitativ beschrieben zu können. Die Kernspinresonanz (NMR)-Spektroskopie ist die beste Methode, um diese Systeme bei atomarer Auflösung zu beobachten und hat bereits viele wertvolle Einsichten gebracht. Wir möchten diese Methode nun mit der Massenspektroskopie (MS) von chemisch verknüpften Peptiden kombinieren, um erweiterte quantitative Modelle zu erhalten. Dazu möchten wir Techniken entwickeln, um die MS-Daten quantitativ interpretieren zu können. Beispielsweise haben verschiedene Peptide unterschiedliche Detektionswahrscheinlichkeiten und wir möchten diese mittels geeigneter Referenzmessungen kalibrieren, um die Menge eines gegeben Peptids exakt bestimmen zu können. Das Ziel unserer Arbeiten ist es, dass wir geeignete Informationen zu Struktur und Dynamik von Chaperon-Substrat-Komplexen aus der Massenspektrometrie erhalten, die wir dann in quantitative Beschreibungen dieser Systeme verwenden können. Im zweiten Teil des Projekts studieren wir die Membranprotein-Insertase YidC. Dieses Protein hat in der Zelle die Funktion, Membranproteine in die Membrane einzusetzen. Dieser Prozess ist biophysikalisch besonders spannend, aber bis jetzt auf atomarer Ebene noch nicht beobachtet worden. Wir werden die insertase YidC und geeignete Substratproteine aufreinigen und ihre Interaktionen, Dynamik und Struktur mithilfe der NMR-Spektroskopie studieren. Moderne hochaufgelöste NMR Methoden erlauben uns, dabei einzelne Aminosäuren zu beobachten und so atomare Auflösung zu erhalten. Unser Ziel wird es sein, den Mechanismus zu verstehen, mit dem YidC seine essentielle Insertasefunktion erfüllt. Wissenschaflicher und gesellschaftlicher Kontext des Forschungsprojekts Unser Projekt befasst sich mit grundlegenden Wissenschaftlichen Fragen aus dem Bereich der Biophysik und Molekularbiologie. Ein mechanistisches Verständnis der Protein- und Membranproteinfaltung ist für das molekulare Verständnis des Lebens unerlässlich. Als praktische Anwendung wird unsere Forschung wird wichtige Beiträge zu Neurodegenerativen Krankheiten machen können. 123 123 OverviewMembersPublicationsProjects & Collaborations
Projects & Collaborations 22 foundShow per page10 10 20 50 Protein-protein interaction antagonists Research Project | 1 Project MembersNo Description available EMBO Fellowship for Patrick Fischer Research Project | 1 Project MembersEMBO Fellowship for Patrick Fischer Structure and mechanism of Leptospira's virulence switch Research Project | 1 Project MembersStructure and mechanism of Leptospira's virulence switch Mechanistic dynamics of the chaperone network in the endoplasmatic reticulum Research Project | 1 Project MembersMechanistic dynamics of the chaperone network in the endoplasmatic reticulum Structure-function analysis of the RNA-binding protein PGC-1a in skeletal muscle Research Project | 2 Project MembersPlasticity of cells, tissues and organs relies on sensing of external and internal cues, integration of the engaged signaling pathways, and orchestration of a pleiotropic response. For example, contractile patterns, ambient temperature and oxygen levels, nutrient availability and composition, as well as other factors result in a massive remodeling of biochemical pathways, cellular metabolism and mechanical properties of skeletal muscle cells, most notably in the context of repeated bouts of exercise, ultimately leading to training adaptations. In turn, such adaptations of skeletal muscle trigger internal and external changes that contribute to the systemic effects of exercise with many health benefits, and strong preventative and therapeutic outcomes in a number of pathologies. Surprisingly, even though the physiological and clinical contributions of exercise-linked muscle plasticity are well recognized, the molecular mechanisms that control the corresponding biological programs are still poorly understood. In recent years, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has emerged as a central regulatory nexus of plasticity in various cell types. PGC-1α integrates upstream signaling pathways to coordinate the complex transcriptional networks encoding biological programs involved in mitochondrial function, cellular metabolism and more. In the case of endurance exercise-mediated plasticity of muscle cells, most, if not all of the adaptations are evoked by this stimulus. Accordingly, muscle-specific overexpression or knockout of PGC-1α elicits high endurance and pathologically sedentary phenotypes, respectively. Our proposal aims at elucidating the molecular underpinnings of the complex transcriptional control that is exerted by PGC-1α in a spatio-temporal manner. We have recently discovered that the binding of RNAs to PGC-1α contributes to full transcriptional activity, and is central for the inclusion of PGC-1α-containing multiprotein complexes in liquid-liquid phase separated nuclear condensates for the sequestration of transcription. We now plan to determine the list and common features of PGC-1α-bound RNAs, interrogate how RNA binding is brought about in a structure-function analysis, study the consequences on multiprotein complex formation, and ultimately investigate the physiological relevance in skeletal muscle cells in vitro and in vivo . To do so, novel animal models will be leveraged for single-end enhanced crosslinking and immunoprecipitation (seCLIP) experiments to identify PGC-1α-bound RNAs, structural information will be obtained by NMR spectroscopy, and validated using site-directed mutagenesis of key amino acids and nucleotides in PGC-1α and RNAs, respectively, to study PGC-1α-dependent liquid-liquid phase separation. Then, PGC-1α-containing multiprotein complexes will be co-purified to perform cryo-electron microscopy-based single particle structural analysis. Finally, the functional consequence of targeted mutagenesis of key residues will be assessed in muscle cells in culture and mouse muscle in vivo with state-of-the-art myotropic adeno-associated viral vectors (AAVMYO). The research plan thus a.) highly synergizes in terms of interdisciplinary approaches between the groups involved and b.) will result in hitherto unprecedented insights into molecular mechanisms that underlie complex spatio-temporal control of transcriptional networks with important implications for the understanding of cellular and tissue plasticity in health and disease. NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria Umbrella Project | 32 Project MembersAntibiotics are powerful and indispensable drugs to treat life threatening bacterial infections such as sepsis or pneumonia. Antibiotics also play a central role in many other areas of modern medicine, in particular to protect patients with compromised immunity during cancer therapies, transplantations or surgical interventions. These achievements are now at risk, with the fraction of bacterial pathogens that are resistant to one or more antibiotics steadily increasing. In addition, development of novel antimicrobials lags behind, suffering from inherently high attrition rates in particular for drug candidates against the most problematic Gram-negative bacteria. Together, these factors increasingly limit the options clinicians have for treating bacterial infections. The overarching goal of NCCR AntiResist is to elucidate the physiological properties of bacterial pathogens in infected human patients in order to find new ways of combatting superbugs. Among the many societal, economic, and scientific factors that impact on the development of alternative strategies for antibiotic discovery, our limited understanding of the physiology and heterogeneity of bacterial pathogens in patients ranks highly. Bacteria growing in tissues of patients experience environments very different from standard laboratory conditions, resulting in radically different microbial physiology and population heterogeneity compared to conditions generally used for antibacterial discovery. There is currently no systematic strategy to overcome this fundamental problem. This has resulted in: (i) suboptimal screens that identify new antibiotics, which do not target the special properties of bacteria growing within the patient; (ii) an inability to properly evaluate the efficacy of non-conventional antibacterial strategies; (iii) missed opportunities for entirely new treatment strategies. This NCCR utilizes patient samples from ongoing clinical studies and establishes a unique multidisciplinary network of clinicians, biologists, engineers, chemists, computational scientists and drug developers that will overcome this problem. We are excited to merge these disciplines in order to determine the properties of pathogens infecting patients, establish conditions in the lab that reproduce these properties and utilize these in-vitro models for antimicrobial discovery and development. In addition, clinical-trial networks and the pharmaceutical industry have major footprints in antimicrobial R&D. Exploiting synergies between these players has great potential for making transformative progress in this critical field of human health. This NCCR maintains active collaborations with Biotech SMEs and large pharmaceutical companies with the goal to: accelerate antibiotic discovery by providing relevant read-outs for early prioritization of compounds; enable innovative screens for non-canonical strategies such as anti-virulence inhibitors and immunomodulators; identify new antibacterial strategies that effectively combat bacteria either by targeting refractory subpopulations or by synergizing with bacterial stresses imposed by the patients' own immune system. This NCCR proposes a paradigm shift in antibiotic discovery by investigating the physiology of bacterial pathogens in human patients. This knowledge will be used to develop assays for molecular analyses and drug screening under relevant conditions and to accelerate antibacterial discovery, improve treatment regimens, and uncover novel targets for eradicating pathogens. Through this concerted effort, this NCCR will make a crucial and unique contribution to winning the race against superbugs. Development of NLRP3-ASC selective inhibitors Research Project | 1 Project MembersDuring an attack from pathogens organisms produce inflammatory cytokines, such as IL-1β and IL-18, that act to combat infection. However, prolonged and uncontrolled production of inflammatory cytokines, usually due to sterile inflammatory triggers, causes chronic inflammation. The latter is the underlying cause of several (auto)-inflammatory diseases such as gout, CAPS (Cryopyrin-Associated Periodic Syndromes), arthritis, Alzheimer's disease, etc. The main cellular node activated by sterile (auto)-inflammatory triggers is NLRP3-inflammasome (NACHT, LRR and PYD domains-containing protein 3). Main drugs against chronic inflammation act downstream of the NLRP3-inflammasome by targeting the release of inflammatory cytokines, with variable efficacity. However, these drugs block the entire inflammatory response, as such, an increase in the rate of infections is a main concern. Alternatively, blocking specifically the NLRP3-inflammasome would inhibit only one branch of the innate immune response having an immense potential to treat (auto)-inflammatory disorders with fewer side effects. As of today, there are no NLRP3-inflamasome targeting drugs on the market. To develop such an inhibitor, we are focusing on 11 small molecules obtained from a cell-based screen of over 10'000 compounds. Our small molecules have novel chemistry to the known NLRP3 inhibitors and, without optimization, show NLRP3-inflammasome inhibitor activity comparable to known NLRP3 inhibitors. During the course of this feasibility study, we will perform hit-to-lead optimization and find the molecular mechanism of action of our compounds. We aim for a business-to-business model. By combining the expertise of our team in structural biology, immunology, organic chemistry and animal experimentation we will generate intellectual property that we can patent, and use as leverage to find business partners to push our compounds towards Phase I clinical trials. A versatile technology platform for identification and development of novel bio-antibiotics Research Project | 1 Project MembersThe rise of multidrug-resistant bacteria threatens the achievements of modern medicine. Particularly problematic in this context is the increasing resistance of the pathogens Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii and Pseudomonas aeruginosa . Antibiotics with novel modes of action are urgently needed to combat these bacterial species. We will establish a technology that enables the identification of high-affinity binders for targets on the surface of these pathogens. These binders are potential novel antibiotics. As a key application, we will target the outer membrane protein insertase BamA. Using the platform, we will select single-domain antibodies, which will be generated either by immunization of alpacas (nanobodies) or in vitro from synthetic nanobody libraries (sybodies), and we will then screen large libraries of such binder molecules for antibiotic activity. We refer to such biomolecular drugs against pathogenic bacteria as "bio-antibiotics". Our technology platform rests on two pillars. In a first step, sybody and nanobody selections are carried out against a purified sample of the target. Notably, only a small subset of the resulting binders will bind the OMP targets in the native context of the bacterial outer membrane, where some accessible epitopes are covered underneath a dense lipopolysaccharide layer. Therefore, in a second step our recently developed binder screening and characterization technology NestLink is applied, which is uniquely available to the Seeger lab. NestLink allows to deep-mine an enriched binder pool in a single experiment, which is impossible by conventional technologies. The utility of our bio-antibiotic technology platform will be demonstrated by the identification of lead molecules that inhibit the growth of clinical Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii and Pseudomonas aeruginosa strains. Suitable lead molecules will enter pre-clinical trials to treat life-threatening blood stream infections and frequently observed urinary tract infections. High-throughput functional assay for BAM-mediated protein folding in a native membrane Research Project | 1 Project MembersFor the development of novel antibiotics against Gram-negative bacteria, targets in the outer membrane or of prime interest. There exist two generally essential such targets, the outer membrane protein insertase BAM with the core unit BamA and the LPS insertion machinery LptD with its associated proteins. Two novel classes of inhibitors for BamA, a synthetic as well as a natural antibiotic, were recently discovered (Luther et al, Nature 2019, Imai et al, Nature 2019). This validity of BAM as a target creates an imperative requirement of an assay to screen for further inhibitors of BAM catalytic activity in vitro . While assays with synthetic vesicles have been reported, so far, no assay for studying BAM-catalyzed OMP folding in the native membrane is available. A quantitative assay to study OMP folding in the natural bacterial membrane has recently been developed by teh Hiller lab in Biozentrum, University of Basel. The assay makes use of outer membrane vesicles (OMVs) that are released from Gram-negative bacteria as spherical buds from the outer membrane, and hence contain the lipid composition and distribution of the outer membrane. They thus provide native and physiological conditions to study the OMP biogenesis mechanism. It is the goal of this collaborative project to establish the assay for high-throughput screening. The assay is expected to allow screening of novel antibiotics, as well as hit validation and lead expansion in high-throughput format. Biophysical Principles of Chaperone-Client Interactions Research Project | 1 Project MembersViele Proteine sind im zellulären Kontext auf die korrekte Entstehung ihrer räumlichen Struktur angewiesen. Diese Struktur können sie oft nur mit Hilfe von anderen Proteinen, sogenannten Chaperonen erreichen. Fehlfaltung und Aggregation bereiten dem Organismus grosse Probleme, die zum Versagen ganzer Organe führen können. Da die Proteinkomplexe aus Chaperonen und ihren Substraten hochdynamisch sind, ist es schwierig, quantitative Beschreibungen davon zu erhalten. Diese sind aber essentiell, um Chaperone genau zu verstehen. Mit unserem Projekt wollen wir zu einem besseren molekularen Verständnis dieser wichtigen Proteinklasse beitragen. Inhalt und Ziele Mit unserem Projekt verfolgen wir zwei Hauptziele. Zum einen möchten wir neue Methoden entwickeln, um die Komplexe aus Chaperonen und Ihren Substraten quantitativ beschrieben zu können. Die Kernspinresonanz (NMR)-Spektroskopie ist die beste Methode, um diese Systeme bei atomarer Auflösung zu beobachten und hat bereits viele wertvolle Einsichten gebracht. Wir möchten diese Methode nun mit der Massenspektroskopie (MS) von chemisch verknüpften Peptiden kombinieren, um erweiterte quantitative Modelle zu erhalten. Dazu möchten wir Techniken entwickeln, um die MS-Daten quantitativ interpretieren zu können. Beispielsweise haben verschiedene Peptide unterschiedliche Detektionswahrscheinlichkeiten und wir möchten diese mittels geeigneter Referenzmessungen kalibrieren, um die Menge eines gegeben Peptids exakt bestimmen zu können. Das Ziel unserer Arbeiten ist es, dass wir geeignete Informationen zu Struktur und Dynamik von Chaperon-Substrat-Komplexen aus der Massenspektrometrie erhalten, die wir dann in quantitative Beschreibungen dieser Systeme verwenden können. Im zweiten Teil des Projekts studieren wir die Membranprotein-Insertase YidC. Dieses Protein hat in der Zelle die Funktion, Membranproteine in die Membrane einzusetzen. Dieser Prozess ist biophysikalisch besonders spannend, aber bis jetzt auf atomarer Ebene noch nicht beobachtet worden. Wir werden die insertase YidC und geeignete Substratproteine aufreinigen und ihre Interaktionen, Dynamik und Struktur mithilfe der NMR-Spektroskopie studieren. Moderne hochaufgelöste NMR Methoden erlauben uns, dabei einzelne Aminosäuren zu beobachten und so atomare Auflösung zu erhalten. Unser Ziel wird es sein, den Mechanismus zu verstehen, mit dem YidC seine essentielle Insertasefunktion erfüllt. Wissenschaflicher und gesellschaftlicher Kontext des Forschungsprojekts Unser Projekt befasst sich mit grundlegenden Wissenschaftlichen Fragen aus dem Bereich der Biophysik und Molekularbiologie. Ein mechanistisches Verständnis der Protein- und Membranproteinfaltung ist für das molekulare Verständnis des Lebens unerlässlich. Als praktische Anwendung wird unsere Forschung wird wichtige Beiträge zu Neurodegenerativen Krankheiten machen können. 123 123
EMBO Fellowship for Patrick Fischer Research Project | 1 Project MembersEMBO Fellowship for Patrick Fischer
Structure and mechanism of Leptospira's virulence switch Research Project | 1 Project MembersStructure and mechanism of Leptospira's virulence switch
Mechanistic dynamics of the chaperone network in the endoplasmatic reticulum Research Project | 1 Project MembersMechanistic dynamics of the chaperone network in the endoplasmatic reticulum
Structure-function analysis of the RNA-binding protein PGC-1a in skeletal muscle Research Project | 2 Project MembersPlasticity of cells, tissues and organs relies on sensing of external and internal cues, integration of the engaged signaling pathways, and orchestration of a pleiotropic response. For example, contractile patterns, ambient temperature and oxygen levels, nutrient availability and composition, as well as other factors result in a massive remodeling of biochemical pathways, cellular metabolism and mechanical properties of skeletal muscle cells, most notably in the context of repeated bouts of exercise, ultimately leading to training adaptations. In turn, such adaptations of skeletal muscle trigger internal and external changes that contribute to the systemic effects of exercise with many health benefits, and strong preventative and therapeutic outcomes in a number of pathologies. Surprisingly, even though the physiological and clinical contributions of exercise-linked muscle plasticity are well recognized, the molecular mechanisms that control the corresponding biological programs are still poorly understood. In recent years, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has emerged as a central regulatory nexus of plasticity in various cell types. PGC-1α integrates upstream signaling pathways to coordinate the complex transcriptional networks encoding biological programs involved in mitochondrial function, cellular metabolism and more. In the case of endurance exercise-mediated plasticity of muscle cells, most, if not all of the adaptations are evoked by this stimulus. Accordingly, muscle-specific overexpression or knockout of PGC-1α elicits high endurance and pathologically sedentary phenotypes, respectively. Our proposal aims at elucidating the molecular underpinnings of the complex transcriptional control that is exerted by PGC-1α in a spatio-temporal manner. We have recently discovered that the binding of RNAs to PGC-1α contributes to full transcriptional activity, and is central for the inclusion of PGC-1α-containing multiprotein complexes in liquid-liquid phase separated nuclear condensates for the sequestration of transcription. We now plan to determine the list and common features of PGC-1α-bound RNAs, interrogate how RNA binding is brought about in a structure-function analysis, study the consequences on multiprotein complex formation, and ultimately investigate the physiological relevance in skeletal muscle cells in vitro and in vivo . To do so, novel animal models will be leveraged for single-end enhanced crosslinking and immunoprecipitation (seCLIP) experiments to identify PGC-1α-bound RNAs, structural information will be obtained by NMR spectroscopy, and validated using site-directed mutagenesis of key amino acids and nucleotides in PGC-1α and RNAs, respectively, to study PGC-1α-dependent liquid-liquid phase separation. Then, PGC-1α-containing multiprotein complexes will be co-purified to perform cryo-electron microscopy-based single particle structural analysis. Finally, the functional consequence of targeted mutagenesis of key residues will be assessed in muscle cells in culture and mouse muscle in vivo with state-of-the-art myotropic adeno-associated viral vectors (AAVMYO). The research plan thus a.) highly synergizes in terms of interdisciplinary approaches between the groups involved and b.) will result in hitherto unprecedented insights into molecular mechanisms that underlie complex spatio-temporal control of transcriptional networks with important implications for the understanding of cellular and tissue plasticity in health and disease.
NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria Umbrella Project | 32 Project MembersAntibiotics are powerful and indispensable drugs to treat life threatening bacterial infections such as sepsis or pneumonia. Antibiotics also play a central role in many other areas of modern medicine, in particular to protect patients with compromised immunity during cancer therapies, transplantations or surgical interventions. These achievements are now at risk, with the fraction of bacterial pathogens that are resistant to one or more antibiotics steadily increasing. In addition, development of novel antimicrobials lags behind, suffering from inherently high attrition rates in particular for drug candidates against the most problematic Gram-negative bacteria. Together, these factors increasingly limit the options clinicians have for treating bacterial infections. The overarching goal of NCCR AntiResist is to elucidate the physiological properties of bacterial pathogens in infected human patients in order to find new ways of combatting superbugs. Among the many societal, economic, and scientific factors that impact on the development of alternative strategies for antibiotic discovery, our limited understanding of the physiology and heterogeneity of bacterial pathogens in patients ranks highly. Bacteria growing in tissues of patients experience environments very different from standard laboratory conditions, resulting in radically different microbial physiology and population heterogeneity compared to conditions generally used for antibacterial discovery. There is currently no systematic strategy to overcome this fundamental problem. This has resulted in: (i) suboptimal screens that identify new antibiotics, which do not target the special properties of bacteria growing within the patient; (ii) an inability to properly evaluate the efficacy of non-conventional antibacterial strategies; (iii) missed opportunities for entirely new treatment strategies. This NCCR utilizes patient samples from ongoing clinical studies and establishes a unique multidisciplinary network of clinicians, biologists, engineers, chemists, computational scientists and drug developers that will overcome this problem. We are excited to merge these disciplines in order to determine the properties of pathogens infecting patients, establish conditions in the lab that reproduce these properties and utilize these in-vitro models for antimicrobial discovery and development. In addition, clinical-trial networks and the pharmaceutical industry have major footprints in antimicrobial R&D. Exploiting synergies between these players has great potential for making transformative progress in this critical field of human health. This NCCR maintains active collaborations with Biotech SMEs and large pharmaceutical companies with the goal to: accelerate antibiotic discovery by providing relevant read-outs for early prioritization of compounds; enable innovative screens for non-canonical strategies such as anti-virulence inhibitors and immunomodulators; identify new antibacterial strategies that effectively combat bacteria either by targeting refractory subpopulations or by synergizing with bacterial stresses imposed by the patients' own immune system. This NCCR proposes a paradigm shift in antibiotic discovery by investigating the physiology of bacterial pathogens in human patients. This knowledge will be used to develop assays for molecular analyses and drug screening under relevant conditions and to accelerate antibacterial discovery, improve treatment regimens, and uncover novel targets for eradicating pathogens. Through this concerted effort, this NCCR will make a crucial and unique contribution to winning the race against superbugs.
Development of NLRP3-ASC selective inhibitors Research Project | 1 Project MembersDuring an attack from pathogens organisms produce inflammatory cytokines, such as IL-1β and IL-18, that act to combat infection. However, prolonged and uncontrolled production of inflammatory cytokines, usually due to sterile inflammatory triggers, causes chronic inflammation. The latter is the underlying cause of several (auto)-inflammatory diseases such as gout, CAPS (Cryopyrin-Associated Periodic Syndromes), arthritis, Alzheimer's disease, etc. The main cellular node activated by sterile (auto)-inflammatory triggers is NLRP3-inflammasome (NACHT, LRR and PYD domains-containing protein 3). Main drugs against chronic inflammation act downstream of the NLRP3-inflammasome by targeting the release of inflammatory cytokines, with variable efficacity. However, these drugs block the entire inflammatory response, as such, an increase in the rate of infections is a main concern. Alternatively, blocking specifically the NLRP3-inflammasome would inhibit only one branch of the innate immune response having an immense potential to treat (auto)-inflammatory disorders with fewer side effects. As of today, there are no NLRP3-inflamasome targeting drugs on the market. To develop such an inhibitor, we are focusing on 11 small molecules obtained from a cell-based screen of over 10'000 compounds. Our small molecules have novel chemistry to the known NLRP3 inhibitors and, without optimization, show NLRP3-inflammasome inhibitor activity comparable to known NLRP3 inhibitors. During the course of this feasibility study, we will perform hit-to-lead optimization and find the molecular mechanism of action of our compounds. We aim for a business-to-business model. By combining the expertise of our team in structural biology, immunology, organic chemistry and animal experimentation we will generate intellectual property that we can patent, and use as leverage to find business partners to push our compounds towards Phase I clinical trials.
A versatile technology platform for identification and development of novel bio-antibiotics Research Project | 1 Project MembersThe rise of multidrug-resistant bacteria threatens the achievements of modern medicine. Particularly problematic in this context is the increasing resistance of the pathogens Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii and Pseudomonas aeruginosa . Antibiotics with novel modes of action are urgently needed to combat these bacterial species. We will establish a technology that enables the identification of high-affinity binders for targets on the surface of these pathogens. These binders are potential novel antibiotics. As a key application, we will target the outer membrane protein insertase BamA. Using the platform, we will select single-domain antibodies, which will be generated either by immunization of alpacas (nanobodies) or in vitro from synthetic nanobody libraries (sybodies), and we will then screen large libraries of such binder molecules for antibiotic activity. We refer to such biomolecular drugs against pathogenic bacteria as "bio-antibiotics". Our technology platform rests on two pillars. In a first step, sybody and nanobody selections are carried out against a purified sample of the target. Notably, only a small subset of the resulting binders will bind the OMP targets in the native context of the bacterial outer membrane, where some accessible epitopes are covered underneath a dense lipopolysaccharide layer. Therefore, in a second step our recently developed binder screening and characterization technology NestLink is applied, which is uniquely available to the Seeger lab. NestLink allows to deep-mine an enriched binder pool in a single experiment, which is impossible by conventional technologies. The utility of our bio-antibiotic technology platform will be demonstrated by the identification of lead molecules that inhibit the growth of clinical Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii and Pseudomonas aeruginosa strains. Suitable lead molecules will enter pre-clinical trials to treat life-threatening blood stream infections and frequently observed urinary tract infections.
High-throughput functional assay for BAM-mediated protein folding in a native membrane Research Project | 1 Project MembersFor the development of novel antibiotics against Gram-negative bacteria, targets in the outer membrane or of prime interest. There exist two generally essential such targets, the outer membrane protein insertase BAM with the core unit BamA and the LPS insertion machinery LptD with its associated proteins. Two novel classes of inhibitors for BamA, a synthetic as well as a natural antibiotic, were recently discovered (Luther et al, Nature 2019, Imai et al, Nature 2019). This validity of BAM as a target creates an imperative requirement of an assay to screen for further inhibitors of BAM catalytic activity in vitro . While assays with synthetic vesicles have been reported, so far, no assay for studying BAM-catalyzed OMP folding in the native membrane is available. A quantitative assay to study OMP folding in the natural bacterial membrane has recently been developed by teh Hiller lab in Biozentrum, University of Basel. The assay makes use of outer membrane vesicles (OMVs) that are released from Gram-negative bacteria as spherical buds from the outer membrane, and hence contain the lipid composition and distribution of the outer membrane. They thus provide native and physiological conditions to study the OMP biogenesis mechanism. It is the goal of this collaborative project to establish the assay for high-throughput screening. The assay is expected to allow screening of novel antibiotics, as well as hit validation and lead expansion in high-throughput format.
Biophysical Principles of Chaperone-Client Interactions Research Project | 1 Project MembersViele Proteine sind im zellulären Kontext auf die korrekte Entstehung ihrer räumlichen Struktur angewiesen. Diese Struktur können sie oft nur mit Hilfe von anderen Proteinen, sogenannten Chaperonen erreichen. Fehlfaltung und Aggregation bereiten dem Organismus grosse Probleme, die zum Versagen ganzer Organe führen können. Da die Proteinkomplexe aus Chaperonen und ihren Substraten hochdynamisch sind, ist es schwierig, quantitative Beschreibungen davon zu erhalten. Diese sind aber essentiell, um Chaperone genau zu verstehen. Mit unserem Projekt wollen wir zu einem besseren molekularen Verständnis dieser wichtigen Proteinklasse beitragen. Inhalt und Ziele Mit unserem Projekt verfolgen wir zwei Hauptziele. Zum einen möchten wir neue Methoden entwickeln, um die Komplexe aus Chaperonen und Ihren Substraten quantitativ beschrieben zu können. Die Kernspinresonanz (NMR)-Spektroskopie ist die beste Methode, um diese Systeme bei atomarer Auflösung zu beobachten und hat bereits viele wertvolle Einsichten gebracht. Wir möchten diese Methode nun mit der Massenspektroskopie (MS) von chemisch verknüpften Peptiden kombinieren, um erweiterte quantitative Modelle zu erhalten. Dazu möchten wir Techniken entwickeln, um die MS-Daten quantitativ interpretieren zu können. Beispielsweise haben verschiedene Peptide unterschiedliche Detektionswahrscheinlichkeiten und wir möchten diese mittels geeigneter Referenzmessungen kalibrieren, um die Menge eines gegeben Peptids exakt bestimmen zu können. Das Ziel unserer Arbeiten ist es, dass wir geeignete Informationen zu Struktur und Dynamik von Chaperon-Substrat-Komplexen aus der Massenspektrometrie erhalten, die wir dann in quantitative Beschreibungen dieser Systeme verwenden können. Im zweiten Teil des Projekts studieren wir die Membranprotein-Insertase YidC. Dieses Protein hat in der Zelle die Funktion, Membranproteine in die Membrane einzusetzen. Dieser Prozess ist biophysikalisch besonders spannend, aber bis jetzt auf atomarer Ebene noch nicht beobachtet worden. Wir werden die insertase YidC und geeignete Substratproteine aufreinigen und ihre Interaktionen, Dynamik und Struktur mithilfe der NMR-Spektroskopie studieren. Moderne hochaufgelöste NMR Methoden erlauben uns, dabei einzelne Aminosäuren zu beobachten und so atomare Auflösung zu erhalten. Unser Ziel wird es sein, den Mechanismus zu verstehen, mit dem YidC seine essentielle Insertasefunktion erfüllt. Wissenschaflicher und gesellschaftlicher Kontext des Forschungsprojekts Unser Projekt befasst sich mit grundlegenden Wissenschaftlichen Fragen aus dem Bereich der Biophysik und Molekularbiologie. Ein mechanistisches Verständnis der Protein- und Membranproteinfaltung ist für das molekulare Verständnis des Lebens unerlässlich. Als praktische Anwendung wird unsere Forschung wird wichtige Beiträge zu Neurodegenerativen Krankheiten machen können.
