Project acronym Autophagy in vitro
Project Reconstituting Autophagosome Biogenesis in vitro
Researcher (PI) Thomas Wollert
Host Institution (HI) INSTITUT PASTEUR
Call Details Starting Grant (StG), LS1, ERC-2014-STG
Summary Autophagy is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Cytotoxic stresses and starvation, however, induces the formation of larger autophagosomes that capture cargo unselectively. Autophagosomes are being generated from a cup-shaped precursor membrane, the isolation membrane, which expands to engulf cytoplasmic components. Sealing of this structure gives rise to the double-membrane surrounded autophagosomes. Two interconnected ubiquitin (Ub)-like conjugation systems coordinate the expansion of autophagosomes by conjugating the autophagy related (Atg)-protein Atg8 to the isolation membrane. In an effort to unravel the function of Atg8, we reconstituted the system on model membranes in vitro and found that Atg8 forms together with the Atg12–Atg5-Atg16 complex a membrane scaffold which is required for productive autophagy in yeast. Humans possess seven Atg8-homologs and two mutually exclusive Atg16-variants. Here, we propose to investigate the function of the human Ub-like conjugation system using a fully reconstituted in vitro system. The spatiotemporal organization of recombinant fluorescent-labeled proteins with synthetic model membranes will be investigated using confocal and TIRF-microscopy. Structural information will be obtained by atomic force and electron microscopy. Mechanistic insights, obtained from the in vitro work, will be tested in vivo in cultured human cells. We belief that revealing 1) the function of the human Ub-like conjugation system in autophagy, 2) the functional differences of Atg8-homologs and the two Atg16-variants Atg16L1 and TECPR1 and 3) how Atg16L1 coordinates non-canonical autophagy will provide essential insights into the pathophysiology of cancer, neurodegenerative, and autoimmune diseases.
Summary
Autophagy is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Cytotoxic stresses and starvation, however, induces the formation of larger autophagosomes that capture cargo unselectively. Autophagosomes are being generated from a cup-shaped precursor membrane, the isolation membrane, which expands to engulf cytoplasmic components. Sealing of this structure gives rise to the double-membrane surrounded autophagosomes. Two interconnected ubiquitin (Ub)-like conjugation systems coordinate the expansion of autophagosomes by conjugating the autophagy related (Atg)-protein Atg8 to the isolation membrane. In an effort to unravel the function of Atg8, we reconstituted the system on model membranes in vitro and found that Atg8 forms together with the Atg12–Atg5-Atg16 complex a membrane scaffold which is required for productive autophagy in yeast. Humans possess seven Atg8-homologs and two mutually exclusive Atg16-variants. Here, we propose to investigate the function of the human Ub-like conjugation system using a fully reconstituted in vitro system. The spatiotemporal organization of recombinant fluorescent-labeled proteins with synthetic model membranes will be investigated using confocal and TIRF-microscopy. Structural information will be obtained by atomic force and electron microscopy. Mechanistic insights, obtained from the in vitro work, will be tested in vivo in cultured human cells. We belief that revealing 1) the function of the human Ub-like conjugation system in autophagy, 2) the functional differences of Atg8-homologs and the two Atg16-variants Atg16L1 and TECPR1 and 3) how Atg16L1 coordinates non-canonical autophagy will provide essential insights into the pathophysiology of cancer, neurodegenerative, and autoimmune diseases.
Max ERC Funding
1 499 726 €
Duration
Start date: 2015-04-01, End date: 2020-03-31
Project acronym Chap4Resp
Project Catching in action a novel bacterial chaperone for respiratory complexes
Researcher (PI) Irina Gutsche
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary Cellular respiration provides energy to power essential processes of life. Respiratory complexes are macromolecular batteries coupling electron flow through a wire of metal clusters and cofactors with proton transfer across the inner membrane of mitochondria and bacteria. Waste products of these cellular factories are reactive oxygen species causing ageing and diseases. Assembly and maturation mechanisms of respiratory complexes remain enigmatic because of their membrane location, multisubunit composition and cofactor insertion. E. coli Complex I, one of the largest membrane proteins, composed of 14 conserved subunits with 9 Fe/S clusters and a flavin, is a minimal model for its 45-subunit human homologue. When proton pumping by respiratory complexes is affected, bacteria become resistant to antibiotics requiring proton gradient for uptake. Based on the latest genetic data, we realize that the huge E. coli macromolecular cage, the structure of which we recently solved by cryo-electron microscopy (cryoEM), in conjunction with a novel protein cofactor, is a specific chaperone for Fe/S cluster biogenesis and assembly of respiratory complexes. This integrated multidisciplinary project combines cryoEM and other structural, biophysical and spectroscopic techniques, to uncover the functional mechanism of this emerging chaperone. The structural plasticity of the chaperone fuelled by ATP hydrolysis, and its interaction with Fe/S cluster biogenesis systems and the main respiratory complexes as a function of stresses, will be scrutinized to gain quasiatomic insights into the way the chaperone operates on its substrates. A novel technology for synergetic in situ investigation of protein complexes in the bacterial cytoplasm by optical imaging, state-of-the-art cryogenic correlative light and electron microscopy, and subtomogram analysis, will be developed and used to obtain snapshots of the chaperone-substrate interactions in the cellular context.
Summary
Cellular respiration provides energy to power essential processes of life. Respiratory complexes are macromolecular batteries coupling electron flow through a wire of metal clusters and cofactors with proton transfer across the inner membrane of mitochondria and bacteria. Waste products of these cellular factories are reactive oxygen species causing ageing and diseases. Assembly and maturation mechanisms of respiratory complexes remain enigmatic because of their membrane location, multisubunit composition and cofactor insertion. E. coli Complex I, one of the largest membrane proteins, composed of 14 conserved subunits with 9 Fe/S clusters and a flavin, is a minimal model for its 45-subunit human homologue. When proton pumping by respiratory complexes is affected, bacteria become resistant to antibiotics requiring proton gradient for uptake. Based on the latest genetic data, we realize that the huge E. coli macromolecular cage, the structure of which we recently solved by cryo-electron microscopy (cryoEM), in conjunction with a novel protein cofactor, is a specific chaperone for Fe/S cluster biogenesis and assembly of respiratory complexes. This integrated multidisciplinary project combines cryoEM and other structural, biophysical and spectroscopic techniques, to uncover the functional mechanism of this emerging chaperone. The structural plasticity of the chaperone fuelled by ATP hydrolysis, and its interaction with Fe/S cluster biogenesis systems and the main respiratory complexes as a function of stresses, will be scrutinized to gain quasiatomic insights into the way the chaperone operates on its substrates. A novel technology for synergetic in situ investigation of protein complexes in the bacterial cytoplasm by optical imaging, state-of-the-art cryogenic correlative light and electron microscopy, and subtomogram analysis, will be developed and used to obtain snapshots of the chaperone-substrate interactions in the cellular context.
Max ERC Funding
1 999 956 €
Duration
Start date: 2015-10-01, End date: 2020-09-30
Project acronym D-END
Project Telomeres: from the DNA end replication problem to the control of cell proliferation
Researcher (PI) Maria Teresa Teixeira Fernandes Bernardo
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2010-StG_20091118
Summary Linear chromosomes of eukaryotes end with telomeres that ensure their stability. Because of the inability of semi-conservative DNA replication machinery to fully replicate DNA ends, telomeres require dedicated mechanisms to be duplicated and their length is eroded at each cell division. For this reason, telomeres constitute molecular clocks that determine cell proliferation potential in eukaryotes. Strikingly, we have shown recently that it is the shortest telomere in the cell that determines the onset of replicative senescence. This project aims a complete and detailed dissection of the in vivo DNA-end replication problem and the deep understanding of its impact for cell division capability. Specifically my goals are (1) the determination of the exact structures that result from the replication of DNA extremities, (2) the examination of the activities operating at the shortest telomere that triggers replicative senescence and (3) the investigation of the correspondence between telomere molecular structure and cell proliferation state at individual cell scale. To achieve this, I will undertake in Saccharomyces cerevisiae original and innovative single-molecule and single-cell approaches, that, in combination with genome-wide screens and sophisticated cellular settings, will allow to track and challenge a specified telomere of defined length. I anticipate that this work will lead to an in-depth understanding of how telomeres are replicated and how they enable the control of cell proliferation in eukaryotic cells, a matter at the intersection of the fundamentals of molecular genetics, cell biology of aging and oncology.
Summary
Linear chromosomes of eukaryotes end with telomeres that ensure their stability. Because of the inability of semi-conservative DNA replication machinery to fully replicate DNA ends, telomeres require dedicated mechanisms to be duplicated and their length is eroded at each cell division. For this reason, telomeres constitute molecular clocks that determine cell proliferation potential in eukaryotes. Strikingly, we have shown recently that it is the shortest telomere in the cell that determines the onset of replicative senescence. This project aims a complete and detailed dissection of the in vivo DNA-end replication problem and the deep understanding of its impact for cell division capability. Specifically my goals are (1) the determination of the exact structures that result from the replication of DNA extremities, (2) the examination of the activities operating at the shortest telomere that triggers replicative senescence and (3) the investigation of the correspondence between telomere molecular structure and cell proliferation state at individual cell scale. To achieve this, I will undertake in Saccharomyces cerevisiae original and innovative single-molecule and single-cell approaches, that, in combination with genome-wide screens and sophisticated cellular settings, will allow to track and challenge a specified telomere of defined length. I anticipate that this work will lead to an in-depth understanding of how telomeres are replicated and how they enable the control of cell proliferation in eukaryotic cells, a matter at the intersection of the fundamentals of molecular genetics, cell biology of aging and oncology.
Max ERC Funding
1 498 504 €
Duration
Start date: 2010-11-01, End date: 2015-10-31
Project acronym DIvA
Project Chromatin function in DNA Double Strand breaks repair: Prime, repair and restore DSB Inducible via AsiSI
Researcher (PI) Gaelle LEGUBE
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary "Among the types of damage, DNA Double Strands Breaks (DSBs) are the most deleterious, as illustrated by the variety of human diseases associated with DSB repair defects. Repair of DSB into the chromatin context raises several questions that we aim to address in this proposal. Firstly, it is likely that the chromatin environment where a break occurs influences the choice of repair pathway. Since the different DSB repair mechanisms can lead to different "scar" on the genome, further studies are required to elucidate how chromatin structure regulates the targeting of DSB repair machineries. Secondly, DNA packaging into chromatin hinders detection and repair of DSBs and many chromatin modifications were recently identified as induced around DSBs to facilitate repair. However, a complete picture of the chromatin landscape set up at DSB, and more specifically the set of histone modifications associated with each repair pathway ("repair histone code") is still awaited. In addition, whether and how damaged chromosomes are reorganized within the nucleus is still unknown. Finally, once repair has been completed, the initial chromatin landscape must be faithfully restored in order to maintain epigenome stability and cell fate.
Using an experimental system we recently developed (called DIvA for DSB Inducible via AsiSI), that allows the induction of multiple sequence-specific DSBs widespread across the genome, we propose to investigate these uncovered aspects of the relationship between chromatin and DSB repair. By high-throughput genomic and proteomic technologies, we will try (i) to understand the contribution of chromatin in the DSB repair pathway choice (PRIME), (ii) to describe more thoroughly the chromatin remodeling events and the spatial chromosomes reorganization, that occur concomitantly to DSB to promote adequate repair (REPAIR), and (iii) to elucidate the processes at work to restore epigenome integrity after DSB repair (RESTORE)."
Summary
"Among the types of damage, DNA Double Strands Breaks (DSBs) are the most deleterious, as illustrated by the variety of human diseases associated with DSB repair defects. Repair of DSB into the chromatin context raises several questions that we aim to address in this proposal. Firstly, it is likely that the chromatin environment where a break occurs influences the choice of repair pathway. Since the different DSB repair mechanisms can lead to different "scar" on the genome, further studies are required to elucidate how chromatin structure regulates the targeting of DSB repair machineries. Secondly, DNA packaging into chromatin hinders detection and repair of DSBs and many chromatin modifications were recently identified as induced around DSBs to facilitate repair. However, a complete picture of the chromatin landscape set up at DSB, and more specifically the set of histone modifications associated with each repair pathway ("repair histone code") is still awaited. In addition, whether and how damaged chromosomes are reorganized within the nucleus is still unknown. Finally, once repair has been completed, the initial chromatin landscape must be faithfully restored in order to maintain epigenome stability and cell fate.
Using an experimental system we recently developed (called DIvA for DSB Inducible via AsiSI), that allows the induction of multiple sequence-specific DSBs widespread across the genome, we propose to investigate these uncovered aspects of the relationship between chromatin and DSB repair. By high-throughput genomic and proteomic technologies, we will try (i) to understand the contribution of chromatin in the DSB repair pathway choice (PRIME), (ii) to describe more thoroughly the chromatin remodeling events and the spatial chromosomes reorganization, that occur concomitantly to DSB to promote adequate repair (REPAIR), and (iii) to elucidate the processes at work to restore epigenome integrity after DSB repair (RESTORE)."
Max ERC Funding
2 000 000 €
Duration
Start date: 2015-07-01, End date: 2020-06-30
Project acronym PentaBrain
Project Structural studies of mammalian Cys-loop receptors
Researcher (PI) Hugues Joseph Nury
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2014-STG
Summary In the brain, Cys-loop receptors mediate fast neurotransmission. They function as allosteric signal transducers across the plasma membrane: upon binding of one or more neurotransmitter molecules to an extracellular site, the receptors undergo complex conformational transitions that result in transient opening of an intrinsic ion channel. The Cys-loop family comprises receptors activated by serotonin, acetylcholine, glycine and GABA. Mammalian receptors are also the targets of a legion of psycho-active and therapeutic compounds (including nicotine, benzodiazepines, anti-emetics, general anaesthetics). Our structural knowledge is currently limited to invertebrate homologues. Atomic structures mammalian receptors are therefore acutely missing in order to understand their physiological role in molecular terms, and to be able to develop new drugs targeting them.
The project proposes to decipher the operation mechanism, the pharmacology and conformational transitions of mammalian Cys-loop receptors. Starting with a solid body of preliminary results, we will obtain new high-resolution structures, taking advantage of antibody-based crystallization chaperones. We will try and record for the first time a ‘molecular movie’ of the gating conformational transition in cristallo. On the way, we will also investigate the potential of antibody-based modulators of Cys-loop receptors for biomedical applications.
The applicant has solved in the past the structures of a bacterial Cys-loop receptor and of the mouse serotonin receptor. The proposed research will take place at the CNRS in Grenoble, France, in a very favourable environment for structural biology.
Summary
In the brain, Cys-loop receptors mediate fast neurotransmission. They function as allosteric signal transducers across the plasma membrane: upon binding of one or more neurotransmitter molecules to an extracellular site, the receptors undergo complex conformational transitions that result in transient opening of an intrinsic ion channel. The Cys-loop family comprises receptors activated by serotonin, acetylcholine, glycine and GABA. Mammalian receptors are also the targets of a legion of psycho-active and therapeutic compounds (including nicotine, benzodiazepines, anti-emetics, general anaesthetics). Our structural knowledge is currently limited to invertebrate homologues. Atomic structures mammalian receptors are therefore acutely missing in order to understand their physiological role in molecular terms, and to be able to develop new drugs targeting them.
The project proposes to decipher the operation mechanism, the pharmacology and conformational transitions of mammalian Cys-loop receptors. Starting with a solid body of preliminary results, we will obtain new high-resolution structures, taking advantage of antibody-based crystallization chaperones. We will try and record for the first time a ‘molecular movie’ of the gating conformational transition in cristallo. On the way, we will also investigate the potential of antibody-based modulators of Cys-loop receptors for biomedical applications.
The applicant has solved in the past the structures of a bacterial Cys-loop receptor and of the mouse serotonin receptor. The proposed research will take place at the CNRS in Grenoble, France, in a very favourable environment for structural biology.
Max ERC Funding
1 500 000 €
Duration
Start date: 2015-06-01, End date: 2020-05-31
Project acronym SEENANOLIFEINACTION
Project Real-Time Studies of Biological NanoMachines in Action by NMR
Researcher (PI) Jerome Boisbouvier
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2010-StG_20091118
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Summary
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Max ERC Funding
1 398 392 €
Duration
Start date: 2011-01-01, End date: 2015-12-31
Project acronym VIDOCK
Project 2D Conformal mapping of protein surfaces: applications to VIsualization and DOCKing software
Researcher (PI) Matthieu Olivier Montes
Host Institution (HI) CONSERVATOIRE NATIONAL DES ARTS ET METIERS
Call Details Starting Grant (StG), LS1, ERC-2014-STG
Summary The goals of structural biology include developing a comprehensive understanding of the molecular shapes and forms embraced by biological macromolecules and extending this knowledge to understand how different molecular architectures are used to perform the chemical reactions that are central to life.
Since the first resolution of protein structures by X-ray crystallography and NMR, structural biology seeks to provide this picture of biological phenomena at the molecular and atomic level by analyzing 3D structures.
In the present proposal, we propose to change this paradigm by changing the mode of representation of protein surfaces to 2D maps. That will open new avenues for 1. the development of innovative high-throughput computation of protein interactions and relationships and 2. the emergence of new forms of visualization and analysis of protein structures and properties. We will apply this powerful tool of conformal mapping to structural biology by representing protein surfaces that are complex 3D surfaces in 2D conformal maps that we will call positive conformal maps. We will extend this representation by also generating the 2D conformal maps of the negatives of the 3D surface of the proteins. These positive and negative 2D conformal maps of the surface of proteins will constitute a new representation of the protein surfaces that will be the basis for innovative high-throughput and/or interactive simulation methods, visualization methods and more generally that will give an other insight on the structure of proteins.
The major impact of this proposal lies in the fact that it will at last open the gates of the long awaited proteome docking. Using a simplified representation of protein surfaces will allow to perform faster complete cross docking calculations and create a new classification of the protein structures based on their surficial similarity.
Summary
The goals of structural biology include developing a comprehensive understanding of the molecular shapes and forms embraced by biological macromolecules and extending this knowledge to understand how different molecular architectures are used to perform the chemical reactions that are central to life.
Since the first resolution of protein structures by X-ray crystallography and NMR, structural biology seeks to provide this picture of biological phenomena at the molecular and atomic level by analyzing 3D structures.
In the present proposal, we propose to change this paradigm by changing the mode of representation of protein surfaces to 2D maps. That will open new avenues for 1. the development of innovative high-throughput computation of protein interactions and relationships and 2. the emergence of new forms of visualization and analysis of protein structures and properties. We will apply this powerful tool of conformal mapping to structural biology by representing protein surfaces that are complex 3D surfaces in 2D conformal maps that we will call positive conformal maps. We will extend this representation by also generating the 2D conformal maps of the negatives of the 3D surface of the proteins. These positive and negative 2D conformal maps of the surface of proteins will constitute a new representation of the protein surfaces that will be the basis for innovative high-throughput and/or interactive simulation methods, visualization methods and more generally that will give an other insight on the structure of proteins.
The major impact of this proposal lies in the fact that it will at last open the gates of the long awaited proteome docking. Using a simplified representation of protein surfaces will allow to perform faster complete cross docking calculations and create a new classification of the protein structures based on their surficial similarity.
Max ERC Funding
1 498 791 €
Duration
Start date: 2015-07-01, End date: 2021-06-30
