Final Report Summary - AMYTOX (Amyloid fibril cytotoxicity: new insights from novel approaches)
Amyloidoses are a major threat to human health and are predicted to dominate world health needs in the coming decades. Despite major research efforts in this area, a cure for most amyloid diseases remains a distant prospect. The challenges in finding a therapy result from our lack of understanding of the structure, stability and dynamics of the different protein aggregates that form during amyloid assembly, the difficulties of visualising large complex, dynamic systems in vitro and in intact cells, and our inability, so far, to identify the molecular species that give rise to the cytotoxicity associated with amyloid disease. The goals of this ERC project were to determine:
(i) The structure of fibril ends and how this relates to their dynamic and cytotoxic properties
At the outset of the project, amyloid fibres were thought to be stable, static entities that are the end product of protein aggregation, but do not directly contribute to disease. Using a range of biochemical, biophysical and cell assays, we showed in the AMYTOX programme that this is not the case. Instead, we showed that amyloid fibrils are unexpectedly dynamic, and can shed toxic particles from their ends (Tipping et al., PNAS (2015); Tipping et al., TIBS (2015)). The results suggest that rather than being inert, amyloid fibrils can instead act as a reservoir of toxic species. Stabilising amyloid fibrils may thus open the door to developing therapies for disease. Building on these observations we have developed powerful new methods for screening for small molecules that prevent aggregation using assays in vitro and in vivo (Young et al., Nature Chemistry (2015), Saunders at el., Nature Chemical Biol (2016)). For analysis of fibril structures for our paper, we developed dark field methods to determine fibril mass per length (Iadanza et al., Sci Rep (2016)) and, exploiting a game-changing £17million investment in EM and NMR instruments in the Astbury Centre for Structural Biology at the University of Leeds, our work culminated in the first structure of a beta-2-microglobulin amyloid fibril to date (Iadanza et al., Nature Comms (2018)).
(ii) The nature of protein-protein and protein-membrane interactions in amyloid formation in vitro and in vivo at the level of single molecules
In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of these early interactions, our understanding of these events remained poor. We have used high resolution NMR methods, exploiting our ultrahigh field NMR instruments in the Astbury Centre to identify, for the first time, the interactions that inhibit (Karamanos et al., Mol Cell (2014)) and those that promote fibril formation in all-atom detail. In addition, we have determined the structure of key amyloid intermediates that explain why some proteins are amyloidogenic, while proteins that are closely related in sequence are not (Karamanos et al., JACS (2016)). These exciting results are now being used in on-going experiments to develop small molecules or other agents able to prevent aggregation by targeting these key species. In parallel with these experiments we have used NMR combined with dynamic force spectroscopy to map the surfaces that initiate amyloid formation in alpha-synuclein - the causative agent of Parkinson’s disease. These single molecule experiments have also revealed specific surfaces essential for controlling aggregation that we hope may lead to new routes to combat amyloidosis.
(iii) The mechanism(s) by which amyloid fibrils kill cells
At the outset of the project we had shown that amyloid fibrils are cytotoxic, as indicated by commonly used assays (MTT reduction). In subsequent experiments, we have now shown that not all fibrils kill cells. However, once fragmented into shorter pieces, amyloid fibrils are readily taken up into endosomes and lysosomes, where they remain as indigestible material that perturbs key functions of these organelles (Jakhria et al., J Biol Chem 2014). We have also shown that fibrils are able to interact with and disrupt membranes, particularly those enriched in lipids found in endosomes and lysosomes (Tipping et al., PNAS (2015)). The susceptibility of fibrils to breakage thus determines their biological effects, and this property is altered by changes in their sequence, including mutations that enhance disease.
(i) The structure of fibril ends and how this relates to their dynamic and cytotoxic properties
At the outset of the project, amyloid fibres were thought to be stable, static entities that are the end product of protein aggregation, but do not directly contribute to disease. Using a range of biochemical, biophysical and cell assays, we showed in the AMYTOX programme that this is not the case. Instead, we showed that amyloid fibrils are unexpectedly dynamic, and can shed toxic particles from their ends (Tipping et al., PNAS (2015); Tipping et al., TIBS (2015)). The results suggest that rather than being inert, amyloid fibrils can instead act as a reservoir of toxic species. Stabilising amyloid fibrils may thus open the door to developing therapies for disease. Building on these observations we have developed powerful new methods for screening for small molecules that prevent aggregation using assays in vitro and in vivo (Young et al., Nature Chemistry (2015), Saunders at el., Nature Chemical Biol (2016)). For analysis of fibril structures for our paper, we developed dark field methods to determine fibril mass per length (Iadanza et al., Sci Rep (2016)) and, exploiting a game-changing £17million investment in EM and NMR instruments in the Astbury Centre for Structural Biology at the University of Leeds, our work culminated in the first structure of a beta-2-microglobulin amyloid fibril to date (Iadanza et al., Nature Comms (2018)).
(ii) The nature of protein-protein and protein-membrane interactions in amyloid formation in vitro and in vivo at the level of single molecules
In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of these early interactions, our understanding of these events remained poor. We have used high resolution NMR methods, exploiting our ultrahigh field NMR instruments in the Astbury Centre to identify, for the first time, the interactions that inhibit (Karamanos et al., Mol Cell (2014)) and those that promote fibril formation in all-atom detail. In addition, we have determined the structure of key amyloid intermediates that explain why some proteins are amyloidogenic, while proteins that are closely related in sequence are not (Karamanos et al., JACS (2016)). These exciting results are now being used in on-going experiments to develop small molecules or other agents able to prevent aggregation by targeting these key species. In parallel with these experiments we have used NMR combined with dynamic force spectroscopy to map the surfaces that initiate amyloid formation in alpha-synuclein - the causative agent of Parkinson’s disease. These single molecule experiments have also revealed specific surfaces essential for controlling aggregation that we hope may lead to new routes to combat amyloidosis.
(iii) The mechanism(s) by which amyloid fibrils kill cells
At the outset of the project we had shown that amyloid fibrils are cytotoxic, as indicated by commonly used assays (MTT reduction). In subsequent experiments, we have now shown that not all fibrils kill cells. However, once fragmented into shorter pieces, amyloid fibrils are readily taken up into endosomes and lysosomes, where they remain as indigestible material that perturbs key functions of these organelles (Jakhria et al., J Biol Chem 2014). We have also shown that fibrils are able to interact with and disrupt membranes, particularly those enriched in lipids found in endosomes and lysosomes (Tipping et al., PNAS (2015)). The susceptibility of fibrils to breakage thus determines their biological effects, and this property is altered by changes in their sequence, including mutations that enhance disease.