Project acronym 20SComplexity
Project An integrative approach to uncover the multilevel regulation of 20S proteasome degradation
Researcher (PI) Michal Sharon
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS1, ERC-2014-STG
Summary For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route.
To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation:
Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome.
Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function.
Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach.
Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.
Summary
For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route.
To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation:
Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome.
Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function.
Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach.
Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.
Max ERC Funding
1 500 000 €
Duration
Start date: 2015-04-01, End date: 2020-03-31
Project acronym 3D-FABRIC
Project 3D Flow Analysis in Bijels Reconfigured for Interfacial Catalysis
Researcher (PI) Martin F. HAASE
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Starting Grant (StG), PE8, ERC-2018-STG
Summary The objective of this proposal is to determine the unknown criteria for convective cross-flow in bicontinuous interfacially jammed emulsion gels (bijels). Based on this, we will answer the question: Can continuously operated interfacial catalysis be realized in bijel cross-flow reactors? Demonstrating this potential will introduce a broadly applicable chemical technology, replacing wasteful chemical processes that require organic solvents. We will achieve our objective in three steps:
(a) Control over bijel structure and properties. Bijels will be formed with a selection of functional inorganic colloidal particles. Nanoparticle surface modifications will be developed and extensively characterized. General principles for the parameters determining bijel structures and properties will be established based on confocal and electron microscopy characterization. These principles will enable unprecedented control over bijel formation and will allow for designing desired properties.
(b) Convective flow in bijels. The mechanical strength of bijels will be tailored and measured. With mechanically robust bijels, the influence of size and organization of oil/water channels on convective mass transfer in bijels will be investigated. To this end, a bijel mass transfer apparatus fabricated by 3d-printing of bijel fibers and soft photolithography will be introduced. In conjunction with the following objective, the analysis of convective flows in bijels will facilitate a thorough description of their structure/function relationships.
(c) Biphasic chemical reactions in STrIPS bijel cross-flow reactors. First, continuous extraction in bijels will be realized. Next, conditions to carry out continuously-operated, phase transfer catalysis of well-known model reactions in bijels will be determined. Both processes will be characterized in-situ and in 3-dimensions by confocal microscopy of fluorescent phase transfer reactions in transparent bijels.
Summary
The objective of this proposal is to determine the unknown criteria for convective cross-flow in bicontinuous interfacially jammed emulsion gels (bijels). Based on this, we will answer the question: Can continuously operated interfacial catalysis be realized in bijel cross-flow reactors? Demonstrating this potential will introduce a broadly applicable chemical technology, replacing wasteful chemical processes that require organic solvents. We will achieve our objective in three steps:
(a) Control over bijel structure and properties. Bijels will be formed with a selection of functional inorganic colloidal particles. Nanoparticle surface modifications will be developed and extensively characterized. General principles for the parameters determining bijel structures and properties will be established based on confocal and electron microscopy characterization. These principles will enable unprecedented control over bijel formation and will allow for designing desired properties.
(b) Convective flow in bijels. The mechanical strength of bijels will be tailored and measured. With mechanically robust bijels, the influence of size and organization of oil/water channels on convective mass transfer in bijels will be investigated. To this end, a bijel mass transfer apparatus fabricated by 3d-printing of bijel fibers and soft photolithography will be introduced. In conjunction with the following objective, the analysis of convective flows in bijels will facilitate a thorough description of their structure/function relationships.
(c) Biphasic chemical reactions in STrIPS bijel cross-flow reactors. First, continuous extraction in bijels will be realized. Next, conditions to carry out continuously-operated, phase transfer catalysis of well-known model reactions in bijels will be determined. Both processes will be characterized in-situ and in 3-dimensions by confocal microscopy of fluorescent phase transfer reactions in transparent bijels.
Max ERC Funding
1 905 000 €
Duration
Start date: 2019-06-01, End date: 2024-05-31
Project acronym 3D-FM
Project Taking Force Microscopy into the Third Dimension
Researcher (PI) Tjerk Hendrik Oosterkamp
Host Institution (HI) UNIVERSITEIT LEIDEN
Call Details Starting Grant (StG), PE3, ERC-2007-StG
Summary I propose to pursue two emerging Force Microscopy techniques that allow measuring structural properties below the surface of the specimen. Whereas Force Microscopy (most commonly known under the name AFM) is usually limited to measuring the surface topography and surface properties of a specimen, I will demonstrate that Force Microscopy can achieve true 3D images of the structure of the cell nucleus. In Ultrasound Force Microscopy, an ultrasound wave is launched from below towards the surface of the specimen. After the sound waves interact with structures beneath the surface of the specimen, the local variations in the amplitude and phase shift of the ultrasonic surface motion is collected by the Force Microscopy tip. Previously, measured 2D maps of the surface response have shown that the surface response is sensitive to structures below the surface. In this project I will employ miniature AFM cantilevers and nanotube tips that I have already developed in my lab. This will allow me to quickly acquire many such 2D maps at a much wider range of ultrasound frequencies and from these 2D maps calculate the full 3D structure below the surface. I expect this technique to have a resolving power better than 10 nm in three dimensions as far as 2 microns below the surface. In parallel I will introduce a major improvement to a technique based on Nuclear Magnetic Resonance (NMR). Magnetic Resonance Force Microscopy measures the interaction of a rotating nuclear spin in the field gradient of a magnetic Force Microscopy tip. However, these forces are so small that they pose an enormous challenge. Miniature cantilevers and nanotube tips, in combination with additional innovations in the detection of the cantilever motion, can overcome this problem. I expect to be able to measure the combined signal of 100 proton spins or fewer, which will allow me to measure proton densities with a resolution of 5 nm, but possibly even with atomic resolution.
Summary
I propose to pursue two emerging Force Microscopy techniques that allow measuring structural properties below the surface of the specimen. Whereas Force Microscopy (most commonly known under the name AFM) is usually limited to measuring the surface topography and surface properties of a specimen, I will demonstrate that Force Microscopy can achieve true 3D images of the structure of the cell nucleus. In Ultrasound Force Microscopy, an ultrasound wave is launched from below towards the surface of the specimen. After the sound waves interact with structures beneath the surface of the specimen, the local variations in the amplitude and phase shift of the ultrasonic surface motion is collected by the Force Microscopy tip. Previously, measured 2D maps of the surface response have shown that the surface response is sensitive to structures below the surface. In this project I will employ miniature AFM cantilevers and nanotube tips that I have already developed in my lab. This will allow me to quickly acquire many such 2D maps at a much wider range of ultrasound frequencies and from these 2D maps calculate the full 3D structure below the surface. I expect this technique to have a resolving power better than 10 nm in three dimensions as far as 2 microns below the surface. In parallel I will introduce a major improvement to a technique based on Nuclear Magnetic Resonance (NMR). Magnetic Resonance Force Microscopy measures the interaction of a rotating nuclear spin in the field gradient of a magnetic Force Microscopy tip. However, these forces are so small that they pose an enormous challenge. Miniature cantilevers and nanotube tips, in combination with additional innovations in the detection of the cantilever motion, can overcome this problem. I expect to be able to measure the combined signal of 100 proton spins or fewer, which will allow me to measure proton densities with a resolution of 5 nm, but possibly even with atomic resolution.
Max ERC Funding
1 794 960 €
Duration
Start date: 2008-08-01, End date: 2013-07-31
Project acronym 4C
Project 4C technology: uncovering the multi-dimensional structure of the genome
Researcher (PI) Wouter Leonard De Laat
Host Institution (HI) KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN - KNAW
Call Details Starting Grant (StG), LS2, ERC-2007-StG
Summary The architecture of DNA in the cell nucleus is an emerging epigenetic key contributor to genome function. We recently developed 4C technology, a high-throughput technique that combines state-of-the-art 3C technology with tailored micro-arrays to uniquely allow for an unbiased genome-wide search for DNA loci that interact in the nuclear space. Based on 4C technology, we were the first to provide a comprehensive overview of long-range DNA contacts of selected loci. The data showed that active and inactive chromatin domains contact many distinct regions within and between chromosomes and genes switch long-range DNA contacts in relation to their expression status. 4C technology not only allows investigating the three-dimensional structure of DNA in the nucleus, it also accurately reconstructs at least 10 megabases of the one-dimensional chromosome sequence map around the target sequence. Changes in this physical map as a result of genomic rearrangements are therefore identified by 4C technology. We recently demonstrated that 4C detects deletions, balanced inversions and translocations in patient samples at a resolution (~7kb) that allowed immediate sequencing of the breakpoints. Excitingly, 4C technology therefore offers the first high-resolution genomic approach that can identify both balanced and unbalanced genomic rearrangements. 4C is expected to become an important tool in clinical diagnosis and prognosis. Key objectives of this proposal are: 1. Explore the functional significance of DNA folding in the nucleus by systematically applying 4C technology to differentially expressed gene loci. 2. Adapt 4C technology such that it allows for massive parallel analysis of DNA interactions between regulatory elements and gene promoters. This method would greatly facilitate the identification of functionally relevant DNA elements in the genome. 3. Develop 4C technology into a clinical diagnostic tool for the accurate detection of balanced and unbalanced rearrangements.
Summary
The architecture of DNA in the cell nucleus is an emerging epigenetic key contributor to genome function. We recently developed 4C technology, a high-throughput technique that combines state-of-the-art 3C technology with tailored micro-arrays to uniquely allow for an unbiased genome-wide search for DNA loci that interact in the nuclear space. Based on 4C technology, we were the first to provide a comprehensive overview of long-range DNA contacts of selected loci. The data showed that active and inactive chromatin domains contact many distinct regions within and between chromosomes and genes switch long-range DNA contacts in relation to their expression status. 4C technology not only allows investigating the three-dimensional structure of DNA in the nucleus, it also accurately reconstructs at least 10 megabases of the one-dimensional chromosome sequence map around the target sequence. Changes in this physical map as a result of genomic rearrangements are therefore identified by 4C technology. We recently demonstrated that 4C detects deletions, balanced inversions and translocations in patient samples at a resolution (~7kb) that allowed immediate sequencing of the breakpoints. Excitingly, 4C technology therefore offers the first high-resolution genomic approach that can identify both balanced and unbalanced genomic rearrangements. 4C is expected to become an important tool in clinical diagnosis and prognosis. Key objectives of this proposal are: 1. Explore the functional significance of DNA folding in the nucleus by systematically applying 4C technology to differentially expressed gene loci. 2. Adapt 4C technology such that it allows for massive parallel analysis of DNA interactions between regulatory elements and gene promoters. This method would greatly facilitate the identification of functionally relevant DNA elements in the genome. 3. Develop 4C technology into a clinical diagnostic tool for the accurate detection of balanced and unbalanced rearrangements.
Max ERC Funding
1 225 000 €
Duration
Start date: 2008-09-01, End date: 2013-08-31
Project acronym 5D-NanoTrack
Project Five-Dimensional Localization Microscopy for Sub-Cellular Dynamics
Researcher (PI) Yoav SHECHTMAN
Host Institution (HI) TECHNION - ISRAEL INSTITUTE OF TECHNOLOGY
Call Details Starting Grant (StG), PE7, ERC-2018-STG
Summary The sub-cellular processes that control the most critical aspects of life occur in three-dimensions (3D), and are intrinsically dynamic. While super-resolution microscopy has revolutionized cellular imaging in recent years, our current capability to observe the dynamics of life on the nanoscale is still extremely limited, due to inherent trade-offs between spatial, temporal and spectral resolution using existing approaches.
We propose to develop and demonstrate an optical microscopy methodology that would enable live sub-cellular observation in unprecedented detail. Making use of multicolor 3D point-spread-function (PSF) engineering, a technique I have recently developed, we will be able to simultaneously track multiple markers inside live cells, at high speed and in five-dimensions (3D, time, and color).
Multicolor 3D PSF engineering holds the potential of being a uniquely powerful method for 5D tracking. However, it is not yet applicable to live-cell imaging, due to significant bottlenecks in optical engineering and signal processing, which we plan to overcome in this project. Importantly, we will also demonstrate the efficacy of our method using a challenging biological application: real-time visualization of chromatin dynamics - the spatiotemporal organization of DNA. This is a highly suitable problem due to its fundamental importance, its role in a variety of cellular processes, and the lack of appropriate tools for studying it.
The project is divided into 3 aims:
1. Technology development: diffractive-element design for multicolor 3D PSFs.
2. System design: volumetric tracking of dense emitters.
3. Live-cell measurements: chromatin dynamics.
Looking ahead, here we create the imaging tools that pave the way towards the holy grail of chromatin visualization: dynamic observation of the 3D positions of the ~3 billion DNA base-pairs in a live human cell. Beyond that, our results will be applicable to numerous 3D micro/nanoscale tracking applications.
Summary
The sub-cellular processes that control the most critical aspects of life occur in three-dimensions (3D), and are intrinsically dynamic. While super-resolution microscopy has revolutionized cellular imaging in recent years, our current capability to observe the dynamics of life on the nanoscale is still extremely limited, due to inherent trade-offs between spatial, temporal and spectral resolution using existing approaches.
We propose to develop and demonstrate an optical microscopy methodology that would enable live sub-cellular observation in unprecedented detail. Making use of multicolor 3D point-spread-function (PSF) engineering, a technique I have recently developed, we will be able to simultaneously track multiple markers inside live cells, at high speed and in five-dimensions (3D, time, and color).
Multicolor 3D PSF engineering holds the potential of being a uniquely powerful method for 5D tracking. However, it is not yet applicable to live-cell imaging, due to significant bottlenecks in optical engineering and signal processing, which we plan to overcome in this project. Importantly, we will also demonstrate the efficacy of our method using a challenging biological application: real-time visualization of chromatin dynamics - the spatiotemporal organization of DNA. This is a highly suitable problem due to its fundamental importance, its role in a variety of cellular processes, and the lack of appropriate tools for studying it.
The project is divided into 3 aims:
1. Technology development: diffractive-element design for multicolor 3D PSFs.
2. System design: volumetric tracking of dense emitters.
3. Live-cell measurements: chromatin dynamics.
Looking ahead, here we create the imaging tools that pave the way towards the holy grail of chromatin visualization: dynamic observation of the 3D positions of the ~3 billion DNA base-pairs in a live human cell. Beyond that, our results will be applicable to numerous 3D micro/nanoscale tracking applications.
Max ERC Funding
1 802 500 €
Duration
Start date: 2018-11-01, End date: 2023-10-31
Project acronym A-LIFE
Project Absorbing aerosol layers in a changing climate: aging, lifetime and dynamics
Researcher (PI) Bernadett Barbara Weinzierl
Host Institution (HI) UNIVERSITAT WIEN
Call Details Starting Grant (StG), PE10, ERC-2014-STG
Summary Aerosols (i.e. tiny particles suspended in the air) are regularly transported in huge amounts over long distances impacting air quality, health, weather and climate thousands of kilometers downwind of the source. Aerosols affect the atmospheric radiation budget through scattering and absorption of solar radiation and through their role as cloud/ice nuclei.
In particular, light absorption by aerosol particles such as mineral dust and black carbon (BC; thought to be the second strongest contribution to current global warming after CO2) is of fundamental importance from a climate perspective because the presence of absorbing particles (1) contributes to solar radiative forcing, (2) heats absorbing aerosol layers, (3) can evaporate clouds and (4) change atmospheric dynamics.
Considering this prominent role of aerosols, vertically-resolved in-situ data on absorbing aerosols are surprisingly scarce and aerosol-dynamic interactions are poorly understood in general. This is, as recognized in the last IPCC report, a serious barrier for taking the accuracy of climate models and predictions to the next level. To overcome this barrier, I propose to investigate aging, lifetime and dynamics of absorbing aerosol layers with a holistic end-to-end approach including laboratory studies, airborne field experiments and numerical model simulations.
Building on the internationally recognized results of my aerosol research group and my long-term experience with airborne aerosol measurements, the time seems ripe to systematically bridge the gap between in-situ measurements of aerosol microphysical and optical properties and the assessment of dynamical interactions of absorbing particles with aerosol layer lifetime through model simulations.
The outcomes of this project will provide fundamental new understanding of absorbing aerosol layers in the climate system and important information for addressing the benefits of BC emission controls for mitigating climate change.
Summary
Aerosols (i.e. tiny particles suspended in the air) are regularly transported in huge amounts over long distances impacting air quality, health, weather and climate thousands of kilometers downwind of the source. Aerosols affect the atmospheric radiation budget through scattering and absorption of solar radiation and through their role as cloud/ice nuclei.
In particular, light absorption by aerosol particles such as mineral dust and black carbon (BC; thought to be the second strongest contribution to current global warming after CO2) is of fundamental importance from a climate perspective because the presence of absorbing particles (1) contributes to solar radiative forcing, (2) heats absorbing aerosol layers, (3) can evaporate clouds and (4) change atmospheric dynamics.
Considering this prominent role of aerosols, vertically-resolved in-situ data on absorbing aerosols are surprisingly scarce and aerosol-dynamic interactions are poorly understood in general. This is, as recognized in the last IPCC report, a serious barrier for taking the accuracy of climate models and predictions to the next level. To overcome this barrier, I propose to investigate aging, lifetime and dynamics of absorbing aerosol layers with a holistic end-to-end approach including laboratory studies, airborne field experiments and numerical model simulations.
Building on the internationally recognized results of my aerosol research group and my long-term experience with airborne aerosol measurements, the time seems ripe to systematically bridge the gap between in-situ measurements of aerosol microphysical and optical properties and the assessment of dynamical interactions of absorbing particles with aerosol layer lifetime through model simulations.
The outcomes of this project will provide fundamental new understanding of absorbing aerosol layers in the climate system and important information for addressing the benefits of BC emission controls for mitigating climate change.
Max ERC Funding
1 987 980 €
Duration
Start date: 2015-10-01, End date: 2020-09-30
Project acronym AAATSI
Project Advanced Antenna Architecture for THZ Sensing Instruments
Researcher (PI) Andrea Neto
Host Institution (HI) TECHNISCHE UNIVERSITEIT DELFT
Call Details Starting Grant (StG), PE7, ERC-2011-StG_20101014
Summary The Tera-Hertz portion of the spectrum presents unique potentials for advanced applications. Currently the THz spectrum is revealing the mechanisms at the origin of our universe and provides the means to monitor the health of our planet via satellite based sensing of critical gases. Potentially time domain sensing of the THz spectrum will be the ideal tool for a vast variety of medical and security applications.
Presently, systems in the THz regime are extremely expensive and consequently the THz spectrum is still the domain of only niche (expensive) scientific applications. The main problems are the lack of power and sensitivity. The wide unused THz spectral bandwidth is, herself, the only widely available resource that in the future can compensate for these problems. But, so far, when scientists try to really use the bandwidth, they run into an insurmountable physical limit: antenna dispersion. Antenna dispersion modifies the signal’s spectrum in a wavelength dependent manner in all types of radiation, but is particularly deleterious to THz signals because the spectrum is too wide and with foreseeable technology it cannot be digitized.
The goal of this proposal is to introduce break-through antenna technology that will eliminate the dispersion bottle neck and revolutionize Time Domain sensing and Spectroscopic Space Science. Achieving these goals the project will pole vault THz imaging technology into the 21-th century and develop critically important enabling technologies which will satisfy the electrical engineering needs of the next 30 years and in the long run will enable multi Tera-bit wireless communications.
In order to achieve these goals, I will first build upon two major breakthrough radiation mechanisms that I pioneered: Leaky Lenses and Connected Arrays. Eventually, ultra wide band imaging arrays constituted by thousands of components will be designed on the bases of the new theoretical findings and demonstrated.
Summary
The Tera-Hertz portion of the spectrum presents unique potentials for advanced applications. Currently the THz spectrum is revealing the mechanisms at the origin of our universe and provides the means to monitor the health of our planet via satellite based sensing of critical gases. Potentially time domain sensing of the THz spectrum will be the ideal tool for a vast variety of medical and security applications.
Presently, systems in the THz regime are extremely expensive and consequently the THz spectrum is still the domain of only niche (expensive) scientific applications. The main problems are the lack of power and sensitivity. The wide unused THz spectral bandwidth is, herself, the only widely available resource that in the future can compensate for these problems. But, so far, when scientists try to really use the bandwidth, they run into an insurmountable physical limit: antenna dispersion. Antenna dispersion modifies the signal’s spectrum in a wavelength dependent manner in all types of radiation, but is particularly deleterious to THz signals because the spectrum is too wide and with foreseeable technology it cannot be digitized.
The goal of this proposal is to introduce break-through antenna technology that will eliminate the dispersion bottle neck and revolutionize Time Domain sensing and Spectroscopic Space Science. Achieving these goals the project will pole vault THz imaging technology into the 21-th century and develop critically important enabling technologies which will satisfy the electrical engineering needs of the next 30 years and in the long run will enable multi Tera-bit wireless communications.
In order to achieve these goals, I will first build upon two major breakthrough radiation mechanisms that I pioneered: Leaky Lenses and Connected Arrays. Eventually, ultra wide band imaging arrays constituted by thousands of components will be designed on the bases of the new theoretical findings and demonstrated.
Max ERC Funding
1 499 487 €
Duration
Start date: 2011-11-01, End date: 2017-10-31
Project acronym ABATSYNAPSE
Project Evolution of Alzheimer’s Disease: From dynamics of single synapses to memory loss
Researcher (PI) Inna Slutsky
Host Institution (HI) TEL AVIV UNIVERSITY
Call Details Starting Grant (StG), LS5, ERC-2011-StG_20101109
Summary A persistent challenge in unravelling mechanisms that regulate memory function is how to bridge the gap between inter-molecular dynamics of single proteins, activity of individual synapses and emerging properties of neuronal circuits. The prototype condition of disintegrating neuronal circuits is Alzheimer’s Disease (AD). Since the early time of Alois Alzheimer at the turn of the 20th century, scientists have been searching for a molecular entity that is in the roots of the cognitive deficits. Although diverse lines of evidence suggest that the amyloid-beta peptide (Abeta) plays a central role in synaptic dysfunctions of AD, several key questions remain unresolved. First, endogenous Abeta peptides are secreted by neurons throughout life, but their physiological functions are largely unknown. Second, experience-dependent physiological mechanisms that initiate the changes in Abeta composition in sporadic, the most frequent form of AD, are unidentified. And finally, molecular mechanisms that trigger Abeta-induced synaptic failure and memory decline remain elusive.
To target these questions, I propose to develop an integrative approach to correlate structure and function at the level of single synapses in hippocampal circuits. State-of-the-art techniques will enable the simultaneous real-time visualization of inter-molecular dynamics within signalling complexes and functional synaptic modifications. Utilizing FRET spectroscopy, high-resolution optical imaging, electrophysiology, molecular biology and biochemistry we will determine the casual relationship between ongoing neuronal activity, temporo-spatial dynamics and molecular composition of Abeta, structural rearrangements within the Abeta signalling complexes and plasticity of single synapses and whole networks. The proposed research will elucidate fundamental principles of neuronal circuits function and identify critical steps that initiate primary synaptic dysfunctions at the very early stages of sporadic AD.
Summary
A persistent challenge in unravelling mechanisms that regulate memory function is how to bridge the gap between inter-molecular dynamics of single proteins, activity of individual synapses and emerging properties of neuronal circuits. The prototype condition of disintegrating neuronal circuits is Alzheimer’s Disease (AD). Since the early time of Alois Alzheimer at the turn of the 20th century, scientists have been searching for a molecular entity that is in the roots of the cognitive deficits. Although diverse lines of evidence suggest that the amyloid-beta peptide (Abeta) plays a central role in synaptic dysfunctions of AD, several key questions remain unresolved. First, endogenous Abeta peptides are secreted by neurons throughout life, but their physiological functions are largely unknown. Second, experience-dependent physiological mechanisms that initiate the changes in Abeta composition in sporadic, the most frequent form of AD, are unidentified. And finally, molecular mechanisms that trigger Abeta-induced synaptic failure and memory decline remain elusive.
To target these questions, I propose to develop an integrative approach to correlate structure and function at the level of single synapses in hippocampal circuits. State-of-the-art techniques will enable the simultaneous real-time visualization of inter-molecular dynamics within signalling complexes and functional synaptic modifications. Utilizing FRET spectroscopy, high-resolution optical imaging, electrophysiology, molecular biology and biochemistry we will determine the casual relationship between ongoing neuronal activity, temporo-spatial dynamics and molecular composition of Abeta, structural rearrangements within the Abeta signalling complexes and plasticity of single synapses and whole networks. The proposed research will elucidate fundamental principles of neuronal circuits function and identify critical steps that initiate primary synaptic dysfunctions at the very early stages of sporadic AD.
Max ERC Funding
2 000 000 €
Duration
Start date: 2011-12-01, End date: 2017-09-30
Project acronym ABCTRANSPORT
Project Minimalist multipurpose ATP-binding cassette transporters
Researcher (PI) Dirk Jan Slotboom
Host Institution (HI) RIJKSUNIVERSITEIT GRONINGEN
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters).
The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms.
Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.
Summary
Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters).
The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms.
Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.
Max ERC Funding
1 500 000 €
Duration
Start date: 2012-01-01, End date: 2017-12-31
Project acronym ABDESIGN
Project Computational design of novel protein function in antibodies
Researcher (PI) Sarel-Jacob Fleishman
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS1, ERC-2013-StG
Summary We propose to elucidate the structural design principles of naturally occurring antibody complementarity-determining regions (CDRs) and to computationally design novel antibody functions. Antibodies represent the most versatile known system for molecular recognition. Research has yielded many insights into antibody design principles and promising biotechnological and pharmaceutical applications. Still, our understanding of how CDRs encode specific loop conformations lags far behind our understanding of structure-function relationships in non-immunological scaffolds. Thus, design of antibodies from first principles has not been demonstrated. We propose a computational-experimental strategy to address this challenge. We will: (a) characterize the design principles and sequence elements that rigidify antibody CDRs. Natural antibody loops will be subjected to computational modeling, crystallography, and a combined in vitro evolution and deep-sequencing approach to isolate sequence features that rigidify loop backbones; (b) develop a novel computational-design strategy, which uses the >1000 solved structures of antibodies deposited in structure databases to realistically model CDRs and design them to recognize proteins that have not been co-crystallized with antibodies. For example, we will design novel antibodies targeting insulin, for which clinically useful diagnostics are needed. By accessing much larger sequence/structure spaces than are available to natural immune-system repertoires and experimental methods, computational antibody design could produce higher-specificity and higher-affinity binders, even to challenging targets; and (c) develop new strategies to program conformational change in CDRs, generating, e.g., the first allosteric antibodies. These will allow targeting, in principle, of any molecule, potentially revolutionizing how antibodies are generated for research and medicine, providing new insights on the design principles of protein functional sites.
Summary
We propose to elucidate the structural design principles of naturally occurring antibody complementarity-determining regions (CDRs) and to computationally design novel antibody functions. Antibodies represent the most versatile known system for molecular recognition. Research has yielded many insights into antibody design principles and promising biotechnological and pharmaceutical applications. Still, our understanding of how CDRs encode specific loop conformations lags far behind our understanding of structure-function relationships in non-immunological scaffolds. Thus, design of antibodies from first principles has not been demonstrated. We propose a computational-experimental strategy to address this challenge. We will: (a) characterize the design principles and sequence elements that rigidify antibody CDRs. Natural antibody loops will be subjected to computational modeling, crystallography, and a combined in vitro evolution and deep-sequencing approach to isolate sequence features that rigidify loop backbones; (b) develop a novel computational-design strategy, which uses the >1000 solved structures of antibodies deposited in structure databases to realistically model CDRs and design them to recognize proteins that have not been co-crystallized with antibodies. For example, we will design novel antibodies targeting insulin, for which clinically useful diagnostics are needed. By accessing much larger sequence/structure spaces than are available to natural immune-system repertoires and experimental methods, computational antibody design could produce higher-specificity and higher-affinity binders, even to challenging targets; and (c) develop new strategies to program conformational change in CDRs, generating, e.g., the first allosteric antibodies. These will allow targeting, in principle, of any molecule, potentially revolutionizing how antibodies are generated for research and medicine, providing new insights on the design principles of protein functional sites.
Max ERC Funding
1 499 930 €
Duration
Start date: 2013-09-01, End date: 2018-08-31
