Project acronym ANTIVIRNA
Project Structural and mechanistic studies of RNA-guided and RNA-targeting antiviral defense pathways
Researcher (PI) Martin Jinek
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Starting Grant (StG), LS1, ERC-2013-StG
Summary The evolutionary pressures exerted by viruses on their host cells constitute a major force that drives the evolution of cellular antiviral mechanisms. The proposed research is motivated by our interest in the roles of protein-RNA interactions in both prokaryotic and eukaryotic antiviral pathways and will proceed in two directions. The first project stems from our current work on the CRISPR pathway, a recently discovered RNA-guided adaptive defense mechanism in bacteria and archaea that silences mobile genetic elements such as viruses (bacteriophages) and plasmids. CRISPR systems rely on short RNAs (crRNAs) that associate with CRISPR-associated (Cas) proteins and function as sequence-specific guides in the detection and destruction of invading nucleic acids. To obtain molecular insights into the mechanisms of crRNA-guided interference, we will pursue structural and functional studies of DNA-targeting ribonuceoprotein complexes from type II and III CRISPR systems. Our work will shed light on the function of these systems in microbial pathogenesis and provide a framework for the informed engineering of RNA-guided gene targeting technologies. The second proposed research direction centres on RNA-targeting antiviral strategies employed by the human innate immune system. Here, our work will focus on structural studies of major interferon-induced effector proteins, initially examining the allosteric activation mechanism of RNase L and subsequently focusing on other antiviral nucleases and RNA helicases, as well as mechanisms by which RNA viruses evade the innate immune response of the host. In our investigations, we plan to approach these questions using an integrated strategy combining structural biology, biochemistry and biophysics with cell-based functional studies. Together, our studies will provide fundamental molecular insights into RNA-centred antiviral mechanisms and their impact on human health and disease.
Summary
The evolutionary pressures exerted by viruses on their host cells constitute a major force that drives the evolution of cellular antiviral mechanisms. The proposed research is motivated by our interest in the roles of protein-RNA interactions in both prokaryotic and eukaryotic antiviral pathways and will proceed in two directions. The first project stems from our current work on the CRISPR pathway, a recently discovered RNA-guided adaptive defense mechanism in bacteria and archaea that silences mobile genetic elements such as viruses (bacteriophages) and plasmids. CRISPR systems rely on short RNAs (crRNAs) that associate with CRISPR-associated (Cas) proteins and function as sequence-specific guides in the detection and destruction of invading nucleic acids. To obtain molecular insights into the mechanisms of crRNA-guided interference, we will pursue structural and functional studies of DNA-targeting ribonuceoprotein complexes from type II and III CRISPR systems. Our work will shed light on the function of these systems in microbial pathogenesis and provide a framework for the informed engineering of RNA-guided gene targeting technologies. The second proposed research direction centres on RNA-targeting antiviral strategies employed by the human innate immune system. Here, our work will focus on structural studies of major interferon-induced effector proteins, initially examining the allosteric activation mechanism of RNase L and subsequently focusing on other antiviral nucleases and RNA helicases, as well as mechanisms by which RNA viruses evade the innate immune response of the host. In our investigations, we plan to approach these questions using an integrated strategy combining structural biology, biochemistry and biophysics with cell-based functional studies. Together, our studies will provide fundamental molecular insights into RNA-centred antiviral mechanisms and their impact on human health and disease.
Max ERC Funding
1 467 180 €
Duration
Start date: 2013-11-01, End date: 2018-10-31
Project acronym ANXIETY & COGNITION
Project How anxiety transforms human cognition: an Affective Neuroscience perspective
Researcher (PI) Gilles Roger Charles Pourtois
Host Institution (HI) UNIVERSITEIT GENT
Call Details Starting Grant (StG), SH3, ERC-2007-StG
Summary Anxiety, a state of apprehension or fear, may provoke cognitive or behavioural disorders and eventually lead to serious medical illnesses. The high prevalence of anxiety disorders in our society sharply contrasts with the lack of clear factual knowledge about the corresponding brain mechanisms at the origin of this profound change in the appraisal of the environment. Little is known about how the psychopathological state of anxiety ultimately turns to a medical condition. The core of this proposal is to gain insight in the neural underpinnings of anxiety and disorders related to anxiety using modern human brain-imaging such as scalp EEG and fMRI. I propose to enlighten how anxiety transforms and shapes human cognition and what the neural correlates and time-course of this modulatory effect are. The primary innovation of this project is the systematic use scalp EEG and fMRI in human participants to better understand the neural mechanisms by which anxiety profoundly influences specific cognitive functions, in particular selective attention and decision-making. The goal of this proposal is to precisely determine the exact timing (using scalp EEG), location, size and extent (using fMRI) of anxiety-related modulations on selective attention and decision-making in the human brain. Here I propose to focus on these two specific processes, because they are likely to reveal selective effects of anxiety on human cognition and can thus serve as powerful models to better figure out how anxiety operates in the human brain. Another important aspect of this project is the fact I envision to help bridge the gap in Health Psychology between fundamental research and clinical practice by proposing alternative revalidation strategies for human adult subjects affected by anxiety-related disorders, which could directly exploit the neuro-scientific discoveries generated in this scientific project.
Summary
Anxiety, a state of apprehension or fear, may provoke cognitive or behavioural disorders and eventually lead to serious medical illnesses. The high prevalence of anxiety disorders in our society sharply contrasts with the lack of clear factual knowledge about the corresponding brain mechanisms at the origin of this profound change in the appraisal of the environment. Little is known about how the psychopathological state of anxiety ultimately turns to a medical condition. The core of this proposal is to gain insight in the neural underpinnings of anxiety and disorders related to anxiety using modern human brain-imaging such as scalp EEG and fMRI. I propose to enlighten how anxiety transforms and shapes human cognition and what the neural correlates and time-course of this modulatory effect are. The primary innovation of this project is the systematic use scalp EEG and fMRI in human participants to better understand the neural mechanisms by which anxiety profoundly influences specific cognitive functions, in particular selective attention and decision-making. The goal of this proposal is to precisely determine the exact timing (using scalp EEG), location, size and extent (using fMRI) of anxiety-related modulations on selective attention and decision-making in the human brain. Here I propose to focus on these two specific processes, because they are likely to reveal selective effects of anxiety on human cognition and can thus serve as powerful models to better figure out how anxiety operates in the human brain. Another important aspect of this project is the fact I envision to help bridge the gap in Health Psychology between fundamental research and clinical practice by proposing alternative revalidation strategies for human adult subjects affected by anxiety-related disorders, which could directly exploit the neuro-scientific discoveries generated in this scientific project.
Max ERC Funding
812 986 €
Duration
Start date: 2008-11-01, End date: 2013-10-31
Project acronym AORVM
Project The Effects of Aging on Object Representation in Visual Working Memory
Researcher (PI) James Robert Brockmole
Host Institution (HI) THE UNIVERSITY OF EDINBURGH
Call Details Starting Grant (StG), SH3, ERC-2007-StG
Summary One’s ability to remember visual material such as objects, faces, and spatial locations over a short period of time declines with age. The proposed research will examine whether these deficits are explained by a reduction in visual working memory (VWM) capacity, or an impairment in one’s ability to maintain, or ‘bind’ appropriate associations among pieces of related information. In this project successful binding is operationally defined as the proper recall or recognition of objects that are defined by the conjunction of multiple visual features. While tests of long-term memory have demonstrated that, despite preserved memory for isolated features, older adults have more difficulty remembering conjunctions of features, no research has yet investigated analogous age related binding deficits in VWM. This is a critical oversight because, given the current state of the science, it is unknown whether these deficits are specific to the long-term memory system, or if they originate in VWM. The project interweaves three strands of research that each investigate whether older adults have more difficulty creating, maintaining, and updating bound multi-feature object representations than younger adults. This theoretical program of enquiry will provide insight into the cognitive architecture of VWM and how this system changes with age, and its outcomes will have wide ranging multi-disciplinary applications in applied theory and intervention techniques that may reduce the adverse consequences of aging on memory.
Summary
One’s ability to remember visual material such as objects, faces, and spatial locations over a short period of time declines with age. The proposed research will examine whether these deficits are explained by a reduction in visual working memory (VWM) capacity, or an impairment in one’s ability to maintain, or ‘bind’ appropriate associations among pieces of related information. In this project successful binding is operationally defined as the proper recall or recognition of objects that are defined by the conjunction of multiple visual features. While tests of long-term memory have demonstrated that, despite preserved memory for isolated features, older adults have more difficulty remembering conjunctions of features, no research has yet investigated analogous age related binding deficits in VWM. This is a critical oversight because, given the current state of the science, it is unknown whether these deficits are specific to the long-term memory system, or if they originate in VWM. The project interweaves three strands of research that each investigate whether older adults have more difficulty creating, maintaining, and updating bound multi-feature object representations than younger adults. This theoretical program of enquiry will provide insight into the cognitive architecture of VWM and how this system changes with age, and its outcomes will have wide ranging multi-disciplinary applications in applied theory and intervention techniques that may reduce the adverse consequences of aging on memory.
Max ERC Funding
500 000 €
Duration
Start date: 2008-09-01, End date: 2011-08-31
Project acronym APES
Project Accuracy and precision for molecular solids
Researcher (PI) Jiri KLIMES
Host Institution (HI) UNIVERZITA KARLOVA
Call Details Starting Grant (StG), PE4, ERC-2017-STG
Summary The description of high pressure phases or polymorphism of molecular solids represents a significant scientific challenge both for experiment and theory. Theoretical methods that are currently used struggle to describe the tiny energy differences between different phases. It is the aim of this project to develop a scheme that would allow accurate and reliable predictions of the binding energies of molecular solids and of the energy differences between different phases.
To reach the required accuracy, we will combine the coupled cluster approach, widely used for reference quality calculations for molecules, with the random phase approximation (RPA) within periodic boundary conditions. As I have recently shown, RPA-based approaches are already some of the most accurate and practically usable methods for the description of extended systems. However, reliability is not only a question of accuracy. Reliable data need to be precise, that is, converged with the numerical parameters so that they are reproducible by other researchers.
Reproducibility is already a growing concern in the field. It is likely to become a considerable issue for highly accurate methods as the calculated energies have a stronger dependence on the simulation parameters such as the basis set size. Two main approaches will be explored to assure precision. First, we will develop the so-called asymptotic correction scheme to speed-up the convergence of the correlation energies with the basis set size. Second, we will directly compare the lattice energies from periodic and finite cluster based calculations. Both should yield identical answers, but if and how the agreement can be reached for general system is currently far from being understood for methods such as coupled cluster. Reliable data will allow us to answer some of the open questions regarding the stability of polymorphs and high pressure phases, such as the possibility of existence of high pressure ionic phases of water and ammonia.
Summary
The description of high pressure phases or polymorphism of molecular solids represents a significant scientific challenge both for experiment and theory. Theoretical methods that are currently used struggle to describe the tiny energy differences between different phases. It is the aim of this project to develop a scheme that would allow accurate and reliable predictions of the binding energies of molecular solids and of the energy differences between different phases.
To reach the required accuracy, we will combine the coupled cluster approach, widely used for reference quality calculations for molecules, with the random phase approximation (RPA) within periodic boundary conditions. As I have recently shown, RPA-based approaches are already some of the most accurate and practically usable methods for the description of extended systems. However, reliability is not only a question of accuracy. Reliable data need to be precise, that is, converged with the numerical parameters so that they are reproducible by other researchers.
Reproducibility is already a growing concern in the field. It is likely to become a considerable issue for highly accurate methods as the calculated energies have a stronger dependence on the simulation parameters such as the basis set size. Two main approaches will be explored to assure precision. First, we will develop the so-called asymptotic correction scheme to speed-up the convergence of the correlation energies with the basis set size. Second, we will directly compare the lattice energies from periodic and finite cluster based calculations. Both should yield identical answers, but if and how the agreement can be reached for general system is currently far from being understood for methods such as coupled cluster. Reliable data will allow us to answer some of the open questions regarding the stability of polymorphs and high pressure phases, such as the possibility of existence of high pressure ionic phases of water and ammonia.
Max ERC Funding
924 375 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
Project acronym APHIDHOST
Project Molecular determinants of aphid host range
Researcher (PI) Jorunn Indra Berit Bos
Host Institution (HI) THE JAMES HUTTON INSTITUTE
Call Details Starting Grant (StG), LS9, ERC-2012-StG_20111109
Summary Many aphid species are restricted to one or few host plants, while some aphids, many of which are of agricultural importance, can infest a wide range of plant species. An important observation is that aphids spend a considerable time on nonhost species, where they probe the leaf tissue and secrete saliva, but for unknown reasons are unable to ingest phloem sap. This suggest that aphids, like plant pathogens, interact with nonhost plants at the molecular level, but potentially are not successful in suppressing plant defenses and/or releasing nutrients. To date, however, the plant cellular changes and the involvement of immune response, such as ETI and PTI, in aphid-host and -nonhost interactions remain elusive. The aim of the proposed project is to gain insight into the level of cellular host reprogramming that takes place during aphid-host interactions, the cellular processes involved in aphid nonhost resistance, and the role of aphid effectors in determining host range. We will compare interactions of two economically important aphid species, Myzus persicae (green peach aphid) and Rhopalosiphum padi (bird cherry oat aphid), with host and nonhost plants. We will investigate local changes in plant cellular processes during aphid-host and -nonhost interactions using microscopy and biochemistry approaches. We will apply a comparative transcriptomics approach and functional assays to identify aphid effectors as potential determinants of host range. Herein we will specifically looks for aphids-species specific effectors and those that are expressed in specific host interactions. To gain insight into molecular mechanisms of effector activities we will identify host targets and investigate the contribution of effector-target interactions to host range. The expected outcomes of the project will, in the long term, contribute to the development of novel strategies to control infestations by aphids and potentially other pests and pathogens, thereby improving food security.
Summary
Many aphid species are restricted to one or few host plants, while some aphids, many of which are of agricultural importance, can infest a wide range of plant species. An important observation is that aphids spend a considerable time on nonhost species, where they probe the leaf tissue and secrete saliva, but for unknown reasons are unable to ingest phloem sap. This suggest that aphids, like plant pathogens, interact with nonhost plants at the molecular level, but potentially are not successful in suppressing plant defenses and/or releasing nutrients. To date, however, the plant cellular changes and the involvement of immune response, such as ETI and PTI, in aphid-host and -nonhost interactions remain elusive. The aim of the proposed project is to gain insight into the level of cellular host reprogramming that takes place during aphid-host interactions, the cellular processes involved in aphid nonhost resistance, and the role of aphid effectors in determining host range. We will compare interactions of two economically important aphid species, Myzus persicae (green peach aphid) and Rhopalosiphum padi (bird cherry oat aphid), with host and nonhost plants. We will investigate local changes in plant cellular processes during aphid-host and -nonhost interactions using microscopy and biochemistry approaches. We will apply a comparative transcriptomics approach and functional assays to identify aphid effectors as potential determinants of host range. Herein we will specifically looks for aphids-species specific effectors and those that are expressed in specific host interactions. To gain insight into molecular mechanisms of effector activities we will identify host targets and investigate the contribution of effector-target interactions to host range. The expected outcomes of the project will, in the long term, contribute to the development of novel strategies to control infestations by aphids and potentially other pests and pathogens, thereby improving food security.
Max ERC Funding
1 463 840 €
Duration
Start date: 2013-02-01, End date: 2018-10-31
Project acronym AppSAM
Project A Flexible Platform for the Application of SAM-dependent enzymes
Researcher (PI) Jennifer Nina ANDEXER
Host Institution (HI) ALBERT-LUDWIGS-UNIVERSITAET FREIBURG
Call Details Starting Grant (StG), LS9, ERC-2016-STG
Summary AppSAM will unlock the synthetic capability of S-adenosyl¬methionine (SAM)-dependent methyltransferases and radical SAM enzymes for application in environmentally friendly and fully sustainable reactions. The biotechnological application of these enzymes will provide access to chemo-, regio- and stereoselective methylations and alkylations, as well as to a wide range of complex rearrangement reactions that are currently not possible through traditional approaches. Methylation reactions are of particular interest due to their importance in epigenetics, cancer metabolism and the development of novel pharmaceuticals. As chemical methylation methods often involve toxic compounds and rarely exhibit the desired selectivity and specificity, there is an urgent need for new, environmentally friendly methodologies.
The proposed project will meet these demands by the provision of modular in vitro and in vivo systems that can be tailored to specific applications. In the first phase of AppSAM, efficient in vitro SAM-regeneration systems will be developed for use with methyltransferases as well as radical SAM enzymes. To achieve this aim, enzymes from different biosynthetic pathways will be combined in multi-enzyme cascades; methods from enzyme and reaction engineering will be used for optimisation. The second phase of AppSAM will address the application on a preparative scale. This will include the isolation of pure product from the in vitro systems, reactions using immobilised enzymes and extracts from in vivo productions. In addition to E. coli, the methylotrophic bacterium Methylobacter extorquens AM1 will be used as a host for the in vivo systems. M. extorquens can use C1 building blocks such as methanol as the sole carbon source, thereby initiating the biotechnological methylation process from a green source material and making the process fully sustainable, as well as being compatible with an envisaged “methanol economy”.
Summary
AppSAM will unlock the synthetic capability of S-adenosyl¬methionine (SAM)-dependent methyltransferases and radical SAM enzymes for application in environmentally friendly and fully sustainable reactions. The biotechnological application of these enzymes will provide access to chemo-, regio- and stereoselective methylations and alkylations, as well as to a wide range of complex rearrangement reactions that are currently not possible through traditional approaches. Methylation reactions are of particular interest due to their importance in epigenetics, cancer metabolism and the development of novel pharmaceuticals. As chemical methylation methods often involve toxic compounds and rarely exhibit the desired selectivity and specificity, there is an urgent need for new, environmentally friendly methodologies.
The proposed project will meet these demands by the provision of modular in vitro and in vivo systems that can be tailored to specific applications. In the first phase of AppSAM, efficient in vitro SAM-regeneration systems will be developed for use with methyltransferases as well as radical SAM enzymes. To achieve this aim, enzymes from different biosynthetic pathways will be combined in multi-enzyme cascades; methods from enzyme and reaction engineering will be used for optimisation. The second phase of AppSAM will address the application on a preparative scale. This will include the isolation of pure product from the in vitro systems, reactions using immobilised enzymes and extracts from in vivo productions. In addition to E. coli, the methylotrophic bacterium Methylobacter extorquens AM1 will be used as a host for the in vivo systems. M. extorquens can use C1 building blocks such as methanol as the sole carbon source, thereby initiating the biotechnological methylation process from a green source material and making the process fully sustainable, as well as being compatible with an envisaged “methanol economy”.
Max ERC Funding
1 499 219 €
Duration
Start date: 2017-10-01, End date: 2022-09-30
Project acronym AQUARAMAN
Project Pipet Based Scanning Probe Microscopy Tip-Enhanced Raman Spectroscopy: A Novel Approach for TERS in Liquids
Researcher (PI) Aleix Garcia Guell
Host Institution (HI) ECOLE POLYTECHNIQUE
Call Details Starting Grant (StG), PE4, ERC-2016-STG
Summary Tip-enhanced Raman spectroscopy (TERS) is often described as the most powerful tool for optical characterization of surfaces and their proximities. It combines the intrinsic spatial resolution of scanning probe techniques (AFM or STM) with the chemical information content of vibrational Raman spectroscopy. Capable to reveal surface heterogeneity at the nanoscale, TERS is currently playing a fundamental role in the understanding of interfacial physicochemical processes in key areas of science and technology such as chemistry, biology and material science.
Unfortunately, the undeniable potential of TERS as a label-free tool for nanoscale chemical and structural characterization is, nowadays, limited to air and vacuum environments, with it failing to operate in a reliable and systematic manner in liquid. The reasons are more technical than fundamental, as what is hindering the application of TERS in water is, among other issues, the low stability of the probes and their consistency. Fields of science and technology where the presence of water/electrolyte is unavoidable, such as biology and electrochemistry, remain unexplored with this powerful technique.
We propose a revolutionary approach for TERS in liquids founded on the employment of pipet-based scanning probe microscopy techniques (pb-SPM) as an alternative to AFM and STM. The use of recent but well established pb-SPM brings the opportunity to develop unprecedented pipet-based TERS probes (beyond the classic and limited metallized solid probes from AFM and STM), together with the implementation of ingenious and innovative measures to enhance tip stability, sensitivity and reliability, unattainable with the current techniques.
We will be in possession of a unique nano-spectroscopy platform capable of experiments in liquids, to follow dynamic processes in-situ, addressing fundamental questions and bringing insight into interfacial phenomena spanning from materials science, physics, chemistry and biology.
Summary
Tip-enhanced Raman spectroscopy (TERS) is often described as the most powerful tool for optical characterization of surfaces and their proximities. It combines the intrinsic spatial resolution of scanning probe techniques (AFM or STM) with the chemical information content of vibrational Raman spectroscopy. Capable to reveal surface heterogeneity at the nanoscale, TERS is currently playing a fundamental role in the understanding of interfacial physicochemical processes in key areas of science and technology such as chemistry, biology and material science.
Unfortunately, the undeniable potential of TERS as a label-free tool for nanoscale chemical and structural characterization is, nowadays, limited to air and vacuum environments, with it failing to operate in a reliable and systematic manner in liquid. The reasons are more technical than fundamental, as what is hindering the application of TERS in water is, among other issues, the low stability of the probes and their consistency. Fields of science and technology where the presence of water/electrolyte is unavoidable, such as biology and electrochemistry, remain unexplored with this powerful technique.
We propose a revolutionary approach for TERS in liquids founded on the employment of pipet-based scanning probe microscopy techniques (pb-SPM) as an alternative to AFM and STM. The use of recent but well established pb-SPM brings the opportunity to develop unprecedented pipet-based TERS probes (beyond the classic and limited metallized solid probes from AFM and STM), together with the implementation of ingenious and innovative measures to enhance tip stability, sensitivity and reliability, unattainable with the current techniques.
We will be in possession of a unique nano-spectroscopy platform capable of experiments in liquids, to follow dynamic processes in-situ, addressing fundamental questions and bringing insight into interfacial phenomena spanning from materials science, physics, chemistry and biology.
Max ERC Funding
1 528 442 €
Duration
Start date: 2017-07-01, End date: 2022-06-30
Project acronym ARCID
Project The Role of Arl Proteins in Retinal and other Ciliary Diseases
Researcher (PI) Alfred Wittinghofer
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Advanced Grant (AdG), LS1, ERC-2010-AdG_20100317
Summary Arl (Arf-like) proteins, GTP-binding proteins of the Ras superfamily, are molecular switches that cycle between a GDP-bound inactive and GTP-bound active state. There are 16 members of the Arl subfamily in the human genome whose basic mechanistic function is unknown. The interactome of Arl2/3 includes proteins involved in retinopathies and other ciliary diseases such as Leber¿s Congenital Amaurosis (LCA) and kidney diseases such as nephronophthisis. Arl6 has been found mutated in Bardet Biedl Syndrome, another pleiotropic ciliary disease. In the proposed interdisciplinary project I want to explore the function of the protein network of Arl2/3 and Arl6 by a combination of biochemical, biophysical and structural methods and use the knowledge obtained to probe their function in live cells. As with other subfamily proteins of the Ras superfamily which have been found to mediate similar biological functions I want to derive a basic understanding of the function of Arl proteins and how it relates to the development and function of the ciliary organelle and how they contribute to ciliary diseases. The molecules in the focus of the project are: the GTP-binding proteins Arl2, 3, 6; RP2, an Arl3GAP mutated in Retinitis pigmentosa; Regulators of Arl2 and 3; PDE¿ and HRG4, effectors of Arl2/3, which bind lipidated proteins; RPGR, mutated in Retinitis pigmentosa, an interactor of PDE¿; RPGRIP and RPGRIPL, interactors of RPGR mutated in LCA and other ciliopathies; Nephrocystin, mutated in nephronophthisis, an interactor of RPGRIP and Arl6, mutated in Bardet Biedl Syndrome, and the BBS complex. The working hypothesis is that Arl protein network(s) mediate ciliary transport processes and that the GTP switch cycle of Arl proteins is an important element of regulation of these processes.
Summary
Arl (Arf-like) proteins, GTP-binding proteins of the Ras superfamily, are molecular switches that cycle between a GDP-bound inactive and GTP-bound active state. There are 16 members of the Arl subfamily in the human genome whose basic mechanistic function is unknown. The interactome of Arl2/3 includes proteins involved in retinopathies and other ciliary diseases such as Leber¿s Congenital Amaurosis (LCA) and kidney diseases such as nephronophthisis. Arl6 has been found mutated in Bardet Biedl Syndrome, another pleiotropic ciliary disease. In the proposed interdisciplinary project I want to explore the function of the protein network of Arl2/3 and Arl6 by a combination of biochemical, biophysical and structural methods and use the knowledge obtained to probe their function in live cells. As with other subfamily proteins of the Ras superfamily which have been found to mediate similar biological functions I want to derive a basic understanding of the function of Arl proteins and how it relates to the development and function of the ciliary organelle and how they contribute to ciliary diseases. The molecules in the focus of the project are: the GTP-binding proteins Arl2, 3, 6; RP2, an Arl3GAP mutated in Retinitis pigmentosa; Regulators of Arl2 and 3; PDE¿ and HRG4, effectors of Arl2/3, which bind lipidated proteins; RPGR, mutated in Retinitis pigmentosa, an interactor of PDE¿; RPGRIP and RPGRIPL, interactors of RPGR mutated in LCA and other ciliopathies; Nephrocystin, mutated in nephronophthisis, an interactor of RPGRIP and Arl6, mutated in Bardet Biedl Syndrome, and the BBS complex. The working hypothesis is that Arl protein network(s) mediate ciliary transport processes and that the GTP switch cycle of Arl proteins is an important element of regulation of these processes.
Max ERC Funding
2 434 400 €
Duration
Start date: 2011-04-01, End date: 2016-03-31
Project acronym ARGO
Project The Quest of the Argonautes - from Myth to Reality
Researcher (PI) JOHN VAN DER OOST
Host Institution (HI) WAGENINGEN UNIVERSITY
Call Details Advanced Grant (AdG), LS1, ERC-2018-ADG
Summary Argonaute nucleases are key players of the eukaryotic RNA interference (RNAi) system. Using small RNA guides, these Argonaute (Ago) proteins specifically target complementary RNA molecules, resulting in regulation of a wide range of crucial processes, including chromosome organization, gene expression and anti-virus defence. Since 2010, my research team has studied closely-related prokaryotic Argonaute (pAgo) variants. This has revealed spectacular mechanistic variations: several thermophilic pAgos catalyse DNA-guided cleavage of double stranded DNA, but only at elevated temperatures. Interestingly, a recently discovered mesophilic Argonaute (CbAgo) can generate double strand DNA breaks at moderate temperatures, providing an excellent basis for this ARGO project. In addition, genome analysis has revealed many distantly-related Argonaute variants, often with unique domain architectures. Hence, the currently known Argonaute homologs are just the tip of the iceberg, and the stage is set for making a big leap in the exploration of the Argonaute family. Initially we will dissect the molecular basis of functional and mechanistic features of uncharacterized natural Argonaute variants, both in eukaryotes (the presence of an Ago-like subunit in the Mediator complex, strongly suggests a regulatory role of an elusive non-coding RNA ligand) and in prokaryotes (selected Ago variants possess distinct domains indicating novel functionalities). After their thorough biochemical characterization, I aim at engineering the functionality of the aforementioned CbAgo through an integrated rational & random approach, i.e. by tinkering of domains, and by an unprecedented in vitro laboratory evolution approach. Eventually, natural & synthetic Argonautes will be selected for their exploitation, and used for developing original genome editing applications (from silencing to base editing). Embarking on this ambitious ARGO expedition will lead us to many exciting discoveries.
Summary
Argonaute nucleases are key players of the eukaryotic RNA interference (RNAi) system. Using small RNA guides, these Argonaute (Ago) proteins specifically target complementary RNA molecules, resulting in regulation of a wide range of crucial processes, including chromosome organization, gene expression and anti-virus defence. Since 2010, my research team has studied closely-related prokaryotic Argonaute (pAgo) variants. This has revealed spectacular mechanistic variations: several thermophilic pAgos catalyse DNA-guided cleavage of double stranded DNA, but only at elevated temperatures. Interestingly, a recently discovered mesophilic Argonaute (CbAgo) can generate double strand DNA breaks at moderate temperatures, providing an excellent basis for this ARGO project. In addition, genome analysis has revealed many distantly-related Argonaute variants, often with unique domain architectures. Hence, the currently known Argonaute homologs are just the tip of the iceberg, and the stage is set for making a big leap in the exploration of the Argonaute family. Initially we will dissect the molecular basis of functional and mechanistic features of uncharacterized natural Argonaute variants, both in eukaryotes (the presence of an Ago-like subunit in the Mediator complex, strongly suggests a regulatory role of an elusive non-coding RNA ligand) and in prokaryotes (selected Ago variants possess distinct domains indicating novel functionalities). After their thorough biochemical characterization, I aim at engineering the functionality of the aforementioned CbAgo through an integrated rational & random approach, i.e. by tinkering of domains, and by an unprecedented in vitro laboratory evolution approach. Eventually, natural & synthetic Argonautes will be selected for their exploitation, and used for developing original genome editing applications (from silencing to base editing). Embarking on this ambitious ARGO expedition will lead us to many exciting discoveries.
Max ERC Funding
2 177 158 €
Duration
Start date: 2019-07-01, End date: 2024-06-30
Project acronym ARISYS
Project Engineering an artificial immune system with functional components assembled from prokaryotic parts and modules
Researcher (PI) Víctor De Lorenzo Prieto
Host Institution (HI) AGENCIA ESTATAL CONSEJO SUPERIOR DEINVESTIGACIONES CIENTIFICAS
Call Details Advanced Grant (AdG), LS9, ERC-2012-ADG_20120314
Summary The objective of this project is to overcome current limitations for antibody production that are inherent to the extant immune system of vertebrates. This will be done by creating an all-in-one artificial/synthetic counterpart based exclusively on prokaryotic parts, devices and modules. To this end, ARISYS will exploit design concepts, construction hierarchies and standardization notions that stem from contemporary Synthetic Biology for the assembly and validation of (what we believe is) the most complex artificial biological system ventured thus far. This all-bacterial immune-like system will not only simplify and make affordable the manipulations necessary for antibody generation, but will also permit the application of such binders by themselves or displayed on bacterial cells to biotechnological challenges well beyond therapeutic and health-related uses. The work plan involves the assembly and validation of autonomous functional modules for [i] displaying antibody/affibody (AB) scaffolds attached to the surface of bacterial cells, [ii] conditional diversification of target-binding sequences of the ABs, [iii] contact-dependent activation of gene expression, [iv] reversible bi-stable switches, and [v] clonal selection and amplification of improved binders. These modules composed of stand-alone parts and bearing well defined input/output functions, will be assembled in the genomic chassis of streamlined Escherichia coli and Pseudomonas putida strains. The resulting molecular network will make the ABs expressed and displayed on the cell surface to proceed spontaneously (or at the user's decision) through subsequent cycles of affinity and specificity maturation towards antigens or other targets presented to the bacterial population. In this way, a single, easy-to-handle (albeit heavily engineered) strain will govern all operations that are typically scattered in a multitude of separate methods and apparatuses for AB production.
Summary
The objective of this project is to overcome current limitations for antibody production that are inherent to the extant immune system of vertebrates. This will be done by creating an all-in-one artificial/synthetic counterpart based exclusively on prokaryotic parts, devices and modules. To this end, ARISYS will exploit design concepts, construction hierarchies and standardization notions that stem from contemporary Synthetic Biology for the assembly and validation of (what we believe is) the most complex artificial biological system ventured thus far. This all-bacterial immune-like system will not only simplify and make affordable the manipulations necessary for antibody generation, but will also permit the application of such binders by themselves or displayed on bacterial cells to biotechnological challenges well beyond therapeutic and health-related uses. The work plan involves the assembly and validation of autonomous functional modules for [i] displaying antibody/affibody (AB) scaffolds attached to the surface of bacterial cells, [ii] conditional diversification of target-binding sequences of the ABs, [iii] contact-dependent activation of gene expression, [iv] reversible bi-stable switches, and [v] clonal selection and amplification of improved binders. These modules composed of stand-alone parts and bearing well defined input/output functions, will be assembled in the genomic chassis of streamlined Escherichia coli and Pseudomonas putida strains. The resulting molecular network will make the ABs expressed and displayed on the cell surface to proceed spontaneously (or at the user's decision) through subsequent cycles of affinity and specificity maturation towards antigens or other targets presented to the bacterial population. In this way, a single, easy-to-handle (albeit heavily engineered) strain will govern all operations that are typically scattered in a multitude of separate methods and apparatuses for AB production.
Max ERC Funding
2 422 271 €
Duration
Start date: 2013-05-01, End date: 2019-04-30
