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: 2020-09-30
Project acronym BacNanoMachine
Project Reconstructing the coordinated self-assembly of a bacterial nanomachine
Researcher (PI) Marc Erhardt
Host Institution (HI) HUMBOLDT-UNIVERSITAET ZU BERLIN
Call Details Consolidator Grant (CoG), LS1, ERC-2019-COG
Summary Life has evolved diverse protein machines and bacteria provide many fascinating examples. Despite being unicellular organisms of relatively small size, bacteria produce sophisticated nanomachines with a high degree of self-organization. The motility organelle of bacteria, the flagellum, is a prime example of complex bacterial nanomachines. Flagella are by far the most prominent extracellular structures known in bacteria and made through self-assembly of several dozen different kinds of proteins and thus represents an ideal model system to study sub-cellular compartmentalization and self-organization. The flagellum can function as a macromolecular motility machine only if its many building blocks assemble in a coordinated manner. However, previous studies have focused on phenotypic and genetic analyses, or the characterization of isolated sub-components. Crucially, how bacteria orchestrate the many different cellular processes in time and space in order to construct a functional motility organelle remains enigmatic. The present proposal constitutes a comprehensive research program with the aim to obtain a holistic understanding of the underlying principles that allow bacteria to control and coordinate the simultaneous self-assembly processes of several multi-component nanomachines within a single cell. Towards this goal, we will combine for the first time the visualization of the dynamic self-assembly of individual flagella with quantitative single-cell gene expression analyses, re-engineering of the genetic network and biophysical modeling in order to develop a biophysical model of flagella self-assembly. This novel, integrative approach will allow us to move beyond the classical, descriptive characterization of protein complexes towards an engineering-type understanding of the extraordinarily robust and coordinated assembly of a multi-component molecular machine.
Summary
Life has evolved diverse protein machines and bacteria provide many fascinating examples. Despite being unicellular organisms of relatively small size, bacteria produce sophisticated nanomachines with a high degree of self-organization. The motility organelle of bacteria, the flagellum, is a prime example of complex bacterial nanomachines. Flagella are by far the most prominent extracellular structures known in bacteria and made through self-assembly of several dozen different kinds of proteins and thus represents an ideal model system to study sub-cellular compartmentalization and self-organization. The flagellum can function as a macromolecular motility machine only if its many building blocks assemble in a coordinated manner. However, previous studies have focused on phenotypic and genetic analyses, or the characterization of isolated sub-components. Crucially, how bacteria orchestrate the many different cellular processes in time and space in order to construct a functional motility organelle remains enigmatic. The present proposal constitutes a comprehensive research program with the aim to obtain a holistic understanding of the underlying principles that allow bacteria to control and coordinate the simultaneous self-assembly processes of several multi-component nanomachines within a single cell. Towards this goal, we will combine for the first time the visualization of the dynamic self-assembly of individual flagella with quantitative single-cell gene expression analyses, re-engineering of the genetic network and biophysical modeling in order to develop a biophysical model of flagella self-assembly. This novel, integrative approach will allow us to move beyond the classical, descriptive characterization of protein complexes towards an engineering-type understanding of the extraordinarily robust and coordinated assembly of a multi-component molecular machine.
Max ERC Funding
1 934 950 €
Duration
Start date: 2020-10-01, End date: 2025-09-30
Project acronym BACTERIAL SYRINGES
Project Protein Translocation Through Bacterial Syringes
Researcher (PI) Stefan Raunser
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Consolidator Grant (CoG), LS1, ERC-2013-CoG
Summary "The main objective of this application is to study the molecular basis of cellular infection by bacterial ABC-type toxins (Tc). Tc complexes are important virulence factors of a range of bacteria, including Photorhabdus luminescens and Yersinia pseudotuberculosis that infect insects and humans. Belonging to the class of pore-forming toxins, tripartite Tc complexes perforate the host membrane by forming channels that translocate toxic enzymes into the host.
In our previous cryo-EM work on the P. luminescens Tc complex we discovered that Tcs use a special syringe-like device for cell entry. Building on these results, we now intend to unravel the molecular mechanism through which this unusual and complicated injection system allows membrane permeation and protein translocation. We will use a hybrid approach, including biochemical reconstitution, structural analysis by cryo-EM and X-ray crystallography, fluorescence-based assays and site-directed mutagenesis to provide a comprehensive description of the molecular mechanism of infection at an unprecedented level of molecular detail.
Our results will be paradigmatic for understanding the mechanism of action of ABC-type toxins and will shed new light on the interactions of bacterial pathogens with their hosts."
Summary
"The main objective of this application is to study the molecular basis of cellular infection by bacterial ABC-type toxins (Tc). Tc complexes are important virulence factors of a range of bacteria, including Photorhabdus luminescens and Yersinia pseudotuberculosis that infect insects and humans. Belonging to the class of pore-forming toxins, tripartite Tc complexes perforate the host membrane by forming channels that translocate toxic enzymes into the host.
In our previous cryo-EM work on the P. luminescens Tc complex we discovered that Tcs use a special syringe-like device for cell entry. Building on these results, we now intend to unravel the molecular mechanism through which this unusual and complicated injection system allows membrane permeation and protein translocation. We will use a hybrid approach, including biochemical reconstitution, structural analysis by cryo-EM and X-ray crystallography, fluorescence-based assays and site-directed mutagenesis to provide a comprehensive description of the molecular mechanism of infection at an unprecedented level of molecular detail.
Our results will be paradigmatic for understanding the mechanism of action of ABC-type toxins and will shed new light on the interactions of bacterial pathogens with their hosts."
Max ERC Funding
1 999 992 €
Duration
Start date: 2014-07-01, End date: 2019-06-30
Project acronym BAS-SBBT
Project Bacterial Amyloid Secretion: Structural Biology and Biotechnology.
Researcher (PI) Han Karel Remaut
Host Institution (HI) VIB VZW
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary Curli are functional amyloid fibers that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria. Unlike the protein misfolding and aggregation events seen in pathological amyloid diseases such as Alzheimer’s and Parkinson’s disease, curli are the product of a dedicated protein secretion machinery. Curli formation requires a specialised and mechanistically unique transporter in the bacterial outer membrane, as well as two soluble accessory proteins thought to facilitate the safe guidance of the curli subunits across the periplasm and to coordinate their self-assembly at cell surface.
In this interdisciplinary research program we will study the structural and molecular biology of E. coli curli biosynthesis and address the fundamental questions concerning the molecular processes that allow the spatially and temporally controlled transport and deposition of these pro-amyloidogenic polypeptides. We will structurally unravel the secretion machinery, trap and analyse critical secretion intermediates and through in vitro reconstitution, assemble a minimal, self-sufficient peptide transport and fiber assembly system.
The new insights gained will set the stage for targeted interventions in curli -mediated biofilm formation and this research project will develop a new framework to harness the unique properties found in curli structure and biosynthesis for biotechnological applications as in patterned functionalized nanowires and directed, selective peptide carriers.
Summary
Curli are functional amyloid fibers that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria. Unlike the protein misfolding and aggregation events seen in pathological amyloid diseases such as Alzheimer’s and Parkinson’s disease, curli are the product of a dedicated protein secretion machinery. Curli formation requires a specialised and mechanistically unique transporter in the bacterial outer membrane, as well as two soluble accessory proteins thought to facilitate the safe guidance of the curli subunits across the periplasm and to coordinate their self-assembly at cell surface.
In this interdisciplinary research program we will study the structural and molecular biology of E. coli curli biosynthesis and address the fundamental questions concerning the molecular processes that allow the spatially and temporally controlled transport and deposition of these pro-amyloidogenic polypeptides. We will structurally unravel the secretion machinery, trap and analyse critical secretion intermediates and through in vitro reconstitution, assemble a minimal, self-sufficient peptide transport and fiber assembly system.
The new insights gained will set the stage for targeted interventions in curli -mediated biofilm formation and this research project will develop a new framework to harness the unique properties found in curli structure and biosynthesis for biotechnological applications as in patterned functionalized nanowires and directed, selective peptide carriers.
Max ERC Funding
1 989 489 €
Duration
Start date: 2015-06-01, End date: 2020-05-31
Project acronym BayCellS
Project A Bayesian Framework for Cellular Structural Biology
Researcher (PI) Michael Nilges
Host Institution (HI) INSTITUT PASTEUR
Call Details Advanced Grant (AdG), LS1, ERC-2011-ADG_20110310
Summary The functioning of a single cell or organism is governed by the laws of chemistry and physics. The bridge from biology to chemistry and physics is provided by structural biology: to understand the functioning of a cell, it is necessary to know the atomic structure of macromolecular assemblies, which may contain hundreds of components. To characterise the structures of the increasingly large and often flexible complexes, high resolution structure determination (as was possible for example for the ribosome) will likely stay the exception, and multiple sources of structural data at multiple resolutions are employed. Integrating these data into one consistent picture poses particular difficulties, since data are much more sparse than in high resolution methods, and the data sets from heterogeneous sources are of highly different and unknown quality and may be mutually inconsistent, and that data are in general averaged over large ensembles and long times. Molecular modelling, a crucial element of any structure determination, plays an even more important role in these multi-scale and multi-technique approaches, not only to obtain structures from the data, but also to evaluate their reliability. This proposal is to develop a consistent framework for this highly complex data integration problem, principally based on Bayesian probability theory. Appropriate models for the major types data types used in hybrid approaches will be developed, as well as representations to include structural knowledge for the components of the complexes, at multiple scales. The new methods will be applied to a series of problems with increasing complexity, going from the determination of protein complexes with high resolution information, over low resolution structures based on protein-protein interaction data such as the nuclear pore, to the genome organisation in the nucleus.
Summary
The functioning of a single cell or organism is governed by the laws of chemistry and physics. The bridge from biology to chemistry and physics is provided by structural biology: to understand the functioning of a cell, it is necessary to know the atomic structure of macromolecular assemblies, which may contain hundreds of components. To characterise the structures of the increasingly large and often flexible complexes, high resolution structure determination (as was possible for example for the ribosome) will likely stay the exception, and multiple sources of structural data at multiple resolutions are employed. Integrating these data into one consistent picture poses particular difficulties, since data are much more sparse than in high resolution methods, and the data sets from heterogeneous sources are of highly different and unknown quality and may be mutually inconsistent, and that data are in general averaged over large ensembles and long times. Molecular modelling, a crucial element of any structure determination, plays an even more important role in these multi-scale and multi-technique approaches, not only to obtain structures from the data, but also to evaluate their reliability. This proposal is to develop a consistent framework for this highly complex data integration problem, principally based on Bayesian probability theory. Appropriate models for the major types data types used in hybrid approaches will be developed, as well as representations to include structural knowledge for the components of the complexes, at multiple scales. The new methods will be applied to a series of problems with increasing complexity, going from the determination of protein complexes with high resolution information, over low resolution structures based on protein-protein interaction data such as the nuclear pore, to the genome organisation in the nucleus.
Max ERC Funding
2 130 212 €
Duration
Start date: 2012-05-01, End date: 2017-04-30
Project acronym BENDER
Project BiogENesis and Degradation of Endoplasmic Reticulum proteins
Researcher (PI) Friedrich Förster
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Consolidator Grant (CoG), LS1, ERC-2016-COG
Summary The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes.
I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.
This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.
Summary
The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes.
I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.
This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.
Max ERC Funding
2 496 611 €
Duration
Start date: 2017-04-01, End date: 2022-03-31
Project acronym BFTERRA
Project Biogenesis and Functions of Telomeric Repeat-containing RNA
Researcher (PI) Claus Maria Azzalin
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Starting Grant (StG), LS1, ERC-2009-StG
Summary Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.
Summary
Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.
Max ERC Funding
1 602 600 €
Duration
Start date: 2009-10-01, End date: 2014-09-30
Project acronym BioMatrix
Project Structural Biology of Exopolysaccharide Secretion in Bacterial Biofilms
Researcher (PI) Petya Violinova KRASTEVA
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary Bacterial biofilm formation is a paramount developmental process in both Gram-positive and Gram-negative species and in many pathogens has been associated with processes of horizontal gene transfer, antibiotic resistance development and pathogen persistence. Bacterial biofilms are collaborative sessile macrocolonies embedded in complex extracellular matrix that secures both mechanical resistance and a medium for intercellular exchange.
Biogenesis platforms for the secretion of biofilm matrix components - many of which controlled directly or indirectly by the intracellular second messenger c-di-GMP - are important determinants for biofilm formation and bacterial disease, and therefore present compelling targets for the development of novel therapeutics. During my Ph.D. and post-doctoral work I studied the structure and function of c-di-GMP-sensing protein factors controling extracellular matrix production by DNA-binding at the transcription initiation level or by inside-out signalling mechanisms at the cell envelope, as well as membrane exporters involved directly in downstream matrix component secretion.
Here, I propose to apply my expertise in microbiology, protein science and structural biology to study the structure and function of exopolysaccharide secretion systems in Gram-negative species. Using Pseudomonas aeruginosa, Vibrio spp. and Escherichia coli as model organisms, my team will aim to reveal the global architecture and individual building components of several expolysaccharide-producing protein megacomplexes. We will combine X-ray crystallography, biophysical and biochemical assays, electron microscopy and in cellulo functional studies to provide a comprehensive view of extracellular matrix production that spans the different resolution levels and presents molecular blueprints for the development of novel anti-infectives. Over the last year I have laid the foundation of these studies and have demonstrated the overall feasibility of the project.
Summary
Bacterial biofilm formation is a paramount developmental process in both Gram-positive and Gram-negative species and in many pathogens has been associated with processes of horizontal gene transfer, antibiotic resistance development and pathogen persistence. Bacterial biofilms are collaborative sessile macrocolonies embedded in complex extracellular matrix that secures both mechanical resistance and a medium for intercellular exchange.
Biogenesis platforms for the secretion of biofilm matrix components - many of which controlled directly or indirectly by the intracellular second messenger c-di-GMP - are important determinants for biofilm formation and bacterial disease, and therefore present compelling targets for the development of novel therapeutics. During my Ph.D. and post-doctoral work I studied the structure and function of c-di-GMP-sensing protein factors controling extracellular matrix production by DNA-binding at the transcription initiation level or by inside-out signalling mechanisms at the cell envelope, as well as membrane exporters involved directly in downstream matrix component secretion.
Here, I propose to apply my expertise in microbiology, protein science and structural biology to study the structure and function of exopolysaccharide secretion systems in Gram-negative species. Using Pseudomonas aeruginosa, Vibrio spp. and Escherichia coli as model organisms, my team will aim to reveal the global architecture and individual building components of several expolysaccharide-producing protein megacomplexes. We will combine X-ray crystallography, biophysical and biochemical assays, electron microscopy and in cellulo functional studies to provide a comprehensive view of extracellular matrix production that spans the different resolution levels and presents molecular blueprints for the development of novel anti-infectives. Over the last year I have laid the foundation of these studies and have demonstrated the overall feasibility of the project.
Max ERC Funding
1 499 901 €
Duration
Start date: 2018-08-01, End date: 2023-07-31
Project acronym BIOMEMOS
Project Higher order structure and function of biomembranes
Researcher (PI) Poul Nissen
Host Institution (HI) AARHUS UNIVERSITET
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies
Summary
The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies
Max ERC Funding
2 444 180 €
Duration
Start date: 2010-04-01, End date: 2015-03-31
Project acronym BIRTOACTION
Project From birth to action: regulation of gene expression through transcription complex biogenesis
Researcher (PI) Laszlo Tora
Host Institution (HI) CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET MEDECINE
Call Details Advanced Grant (AdG), LS1, ERC-2013-ADG
Summary "Transcriptional regulation of protein coding genes in eukaryotic cells requires a complex interplay of sequence-specific DNA-binding factors, co-activators, general transcription factors (GTFs), RNA polymerase II and the epigenetic status of target sequences. Nuclear transcription complexes function as large multiprotein assemblies and are often composed of functional modules. The regulated decision-making that exists in cells governing the assembly and the allocation of factors to different transcription complexes to regulate distinct gene expression pathways is not yet understood. To tackle this fundamental question, we will systematically analyse the regulated biogenesis of transcription complexes from their sites of translation in the cytoplasm, through their assembly intermediates and nuclear import, to their site of action in the nucleus. The project will have four main Aims to decipher the biogenesis of transcription complexes:
I) Investigate their co-translation-driven assembly
II) Determine their cytoplasmic intermediates and factors required for their assembly pathways
III) Uncover their nuclear import
IV) Understand at the single molecule level their nuclear assembly, dynamics and action at target genes
To carry out these aims we propose a combination of multidisciplinary and cutting edge approaches, out of which some of them will be high-risk taking, while others will utilize methods routinely run by the group. The project builds on several complementary expertise and knowledge either already existing in the group or that will be implemented during the project. At the end of the proposed project we will obtain novel results extensively describing the different steps of the regulatory mechanisms that control the assembly and the consequent gene regulatory function of transcription complexes. Thus, we anticipate that the results of our research will have a major impact on the field and will lead to a new paradigm for contemporary metazoan transcription."
Summary
"Transcriptional regulation of protein coding genes in eukaryotic cells requires a complex interplay of sequence-specific DNA-binding factors, co-activators, general transcription factors (GTFs), RNA polymerase II and the epigenetic status of target sequences. Nuclear transcription complexes function as large multiprotein assemblies and are often composed of functional modules. The regulated decision-making that exists in cells governing the assembly and the allocation of factors to different transcription complexes to regulate distinct gene expression pathways is not yet understood. To tackle this fundamental question, we will systematically analyse the regulated biogenesis of transcription complexes from their sites of translation in the cytoplasm, through their assembly intermediates and nuclear import, to their site of action in the nucleus. The project will have four main Aims to decipher the biogenesis of transcription complexes:
I) Investigate their co-translation-driven assembly
II) Determine their cytoplasmic intermediates and factors required for their assembly pathways
III) Uncover their nuclear import
IV) Understand at the single molecule level their nuclear assembly, dynamics and action at target genes
To carry out these aims we propose a combination of multidisciplinary and cutting edge approaches, out of which some of them will be high-risk taking, while others will utilize methods routinely run by the group. The project builds on several complementary expertise and knowledge either already existing in the group or that will be implemented during the project. At the end of the proposed project we will obtain novel results extensively describing the different steps of the regulatory mechanisms that control the assembly and the consequent gene regulatory function of transcription complexes. Thus, we anticipate that the results of our research will have a major impact on the field and will lead to a new paradigm for contemporary metazoan transcription."
Max ERC Funding
2 500 000 €
Duration
Start date: 2014-01-01, End date: 2018-12-31
