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 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