Project acronym CilDyn
Project Molecular analysis of the Hedgehog signal transduction complex in the primary cilium
Researcher (PI) Christian Siebold
Host Institution (HI) THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary The unexpected connection between the primary cilium and cell-to-cell signalling is one of the most exciting discoveries in cell and developmental biology in the last decade. In particular, the Hedgehog (Hh) pathway relies on the primary cilium to fulfil its fundamental functions in orchestrating vertebrate development. This microtubule-based antenna, up to 5 µm long, protrudes from the plasma membrane of almost every human cell and is the essential compartment for the entire Hh signalling cascade. All its molecular components, from the most upstream transmembrane Hh receptor down to the ultimate transcription factors, are dynamically localised and enriched in the primary cilium. The aim of this proposal, which combines structural biology and live cell imaging, is to understand the function and signalling consequences of the multivalent interactions between Hh signal transducer proteins as well as their spatial and temporal regulation in the primary cilium. The key questions my laboratory will address are: What are the rules for assembly of Hh signal transduction complexes? How dynamic are these complexes in size and organisation? How are these processes linked to the transport and accumulation in the primary cilium?
I will combine state-of-the art structural biology techniques (with an emphasis on X-ray crystallography) to study the molecular architecture of binary and higher-order Hh signal transduction complexes and live cell fluorescence microscopy (for protein localisation and direct protein interactions). These two approaches will allow me to identify and define specific protein-protein interfaces at the atomic level and test their functional consequences in the cell in real time. My goal is to consolidate a world-class morphogen signal transduction laboratory, deciphering fundamental biological insights. Importantly, my results and reagents can potentially feed into the development of novel anti-cancer therapeutics and reagents promoting stem cell therapy.
Summary
The unexpected connection between the primary cilium and cell-to-cell signalling is one of the most exciting discoveries in cell and developmental biology in the last decade. In particular, the Hedgehog (Hh) pathway relies on the primary cilium to fulfil its fundamental functions in orchestrating vertebrate development. This microtubule-based antenna, up to 5 µm long, protrudes from the plasma membrane of almost every human cell and is the essential compartment for the entire Hh signalling cascade. All its molecular components, from the most upstream transmembrane Hh receptor down to the ultimate transcription factors, are dynamically localised and enriched in the primary cilium. The aim of this proposal, which combines structural biology and live cell imaging, is to understand the function and signalling consequences of the multivalent interactions between Hh signal transducer proteins as well as their spatial and temporal regulation in the primary cilium. The key questions my laboratory will address are: What are the rules for assembly of Hh signal transduction complexes? How dynamic are these complexes in size and organisation? How are these processes linked to the transport and accumulation in the primary cilium?
I will combine state-of-the art structural biology techniques (with an emphasis on X-ray crystallography) to study the molecular architecture of binary and higher-order Hh signal transduction complexes and live cell fluorescence microscopy (for protein localisation and direct protein interactions). These two approaches will allow me to identify and define specific protein-protein interfaces at the atomic level and test their functional consequences in the cell in real time. My goal is to consolidate a world-class morphogen signal transduction laboratory, deciphering fundamental biological insights. Importantly, my results and reagents can potentially feed into the development of novel anti-cancer therapeutics and reagents promoting stem cell therapy.
Max ERC Funding
1 727 456 €
Duration
Start date: 2015-08-01, End date: 2020-07-31
Project acronym MEMBRANEFUSION
Project Structure and mechanism of viral and cellular membrane fusion machineries
Researcher (PI) John Briggs
Host Institution (HI) MEDICAL RESEARCH COUNCIL
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary Fusion of two biological membranes is essential to life. It is required during organism development, for trafficking of material between cellular compartments, for transfer of information across synapses, and for entry of viruses into cells. Fusion must be carefully controlled and the core fusion components are typically found within a complex regulatory machine. There have been decades of research on the structure and function of individual components, on the dynamics and biophysics of fusion, and on phenotypes resulting from mutating or inhibiting component proteins. These have led to a model for fusion in which regulated refolding or assembly of proteins draws two membranes closer together until they fuse. Despite this breadth of study, we know very little about how the components of the fusion machinery function in context: How are they arranged on the membrane around the site of fusion? How do they respond structurally to regulation? How does the fully assembled machinery rearrange to reshape the membrane and drive fusion? These gaps in knowledge can be attributed to a shortage of structural biology methods able to derive structural data on proteins assembled within complex, heterogeneous or dynamic environments such as a fusion site. Here I propose to apply a combination of state-of-the-art cryo-electron tomography, image processing and correlative fluorescence and electron microscopy methods to obtain detailed structural information on assembled fusion machineries and of fusion intermediates both in vitro and in vivo. I will study how influenza virus fuses with a target membrane, complemented by studies on fusion of HIV-1 and of synaptic vesicles. By determining how viral and synaptic fusion complexes reposition and restructure prior to fusion, how they arrange around the fusion site, how they reshape the membrane to induce fusion, and how these processes can be regulated and inhibited, I will derive a mechanistic model of membrane fusion in situ.
Summary
Fusion of two biological membranes is essential to life. It is required during organism development, for trafficking of material between cellular compartments, for transfer of information across synapses, and for entry of viruses into cells. Fusion must be carefully controlled and the core fusion components are typically found within a complex regulatory machine. There have been decades of research on the structure and function of individual components, on the dynamics and biophysics of fusion, and on phenotypes resulting from mutating or inhibiting component proteins. These have led to a model for fusion in which regulated refolding or assembly of proteins draws two membranes closer together until they fuse. Despite this breadth of study, we know very little about how the components of the fusion machinery function in context: How are they arranged on the membrane around the site of fusion? How do they respond structurally to regulation? How does the fully assembled machinery rearrange to reshape the membrane and drive fusion? These gaps in knowledge can be attributed to a shortage of structural biology methods able to derive structural data on proteins assembled within complex, heterogeneous or dynamic environments such as a fusion site. Here I propose to apply a combination of state-of-the-art cryo-electron tomography, image processing and correlative fluorescence and electron microscopy methods to obtain detailed structural information on assembled fusion machineries and of fusion intermediates both in vitro and in vivo. I will study how influenza virus fuses with a target membrane, complemented by studies on fusion of HIV-1 and of synaptic vesicles. By determining how viral and synaptic fusion complexes reposition and restructure prior to fusion, how they arrange around the fusion site, how they reshape the membrane to induce fusion, and how these processes can be regulated and inhibited, I will derive a mechanistic model of membrane fusion in situ.
Max ERC Funding
1 965 961 €
Duration
Start date: 2015-09-01, End date: 2021-08-31
Project acronym RINGE3
Project Structural and mechanistic insights into RING E3-mediated ubiquitination
Researcher (PI) Danny Huang
Host Institution (HI) BEATSON INSTITUTE FOR CANCER RESEARCH LBG
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary Ubiquitin (Ub) conjugation regulates a myriad of cellular processes in the eukaryotic cell. Ub-ligases (E3) play a pivotal role in deciding the substrate’s fate and function by catalyzing the transfer of Ub from Ub-conjugating enzyme (E2) to a substrate protein lysine sidechain. Successive rounds of E3-catalyzed substrate ubiquitination lead to the formation of poly-Ub chains or multi-monoubiquitination, which direct the substrate to different biological fates such as degradation by the 26S proteasome. RING E3s comprise the largest family of E3s with approximately 600 members in humans. Over the last fifteen years, structural biology and biochemical studies have paved the way for understanding how RING E3s interact with E2s and substrates, and how they are regulated. Recently my group has trapped the crystal structure of a RING E3 bound to an E2 covalently-linked to Ub (E2~Ub), thus providing a molecular snapshot of how RING E3 optimizes E2~Ub for catalysis. Despite these advances, the mechanisms of RING E3-catalyzed ubiquitination are not completely understood. Here, we propose to investigate three key aspects of RING E3 functions. First, we will determine structures of several RING E3s bound to E2~Ub to dissect the molecular basis for RING E3-E2~Ub selectivity. Second, our recent structure of a RING E3, Cbl-b, bound to E2~Ub and a substrate peptide provides a starting point for structural determination of a more challenging RING E3-E2~Ub-intact substrate complex to elucidate the mechanisms of substrate ubiquitination. Third, we have developed an ubiquitinated Cbl-substrate mimetic to study the mechanisms of RING E3-catalyzed poly-ubiquitination using structural and biochemical approaches. Expected results will greatly expand our knowledge of RING E3-mediated ubiquitination and will foster strategies in exploiting E3s for therapeutic development, since deregulation of E3s underlies many diseases including cancers.
Summary
Ubiquitin (Ub) conjugation regulates a myriad of cellular processes in the eukaryotic cell. Ub-ligases (E3) play a pivotal role in deciding the substrate’s fate and function by catalyzing the transfer of Ub from Ub-conjugating enzyme (E2) to a substrate protein lysine sidechain. Successive rounds of E3-catalyzed substrate ubiquitination lead to the formation of poly-Ub chains or multi-monoubiquitination, which direct the substrate to different biological fates such as degradation by the 26S proteasome. RING E3s comprise the largest family of E3s with approximately 600 members in humans. Over the last fifteen years, structural biology and biochemical studies have paved the way for understanding how RING E3s interact with E2s and substrates, and how they are regulated. Recently my group has trapped the crystal structure of a RING E3 bound to an E2 covalently-linked to Ub (E2~Ub), thus providing a molecular snapshot of how RING E3 optimizes E2~Ub for catalysis. Despite these advances, the mechanisms of RING E3-catalyzed ubiquitination are not completely understood. Here, we propose to investigate three key aspects of RING E3 functions. First, we will determine structures of several RING E3s bound to E2~Ub to dissect the molecular basis for RING E3-E2~Ub selectivity. Second, our recent structure of a RING E3, Cbl-b, bound to E2~Ub and a substrate peptide provides a starting point for structural determination of a more challenging RING E3-E2~Ub-intact substrate complex to elucidate the mechanisms of substrate ubiquitination. Third, we have developed an ubiquitinated Cbl-substrate mimetic to study the mechanisms of RING E3-catalyzed poly-ubiquitination using structural and biochemical approaches. Expected results will greatly expand our knowledge of RING E3-mediated ubiquitination and will foster strategies in exploiting E3s for therapeutic development, since deregulation of E3s underlies many diseases including cancers.
Max ERC Funding
2 000 000 €
Duration
Start date: 2015-05-01, End date: 2020-04-30
