Defining the molecular pathway for yeast prion fibril assembly using cryo-electron microscopy

Inherited traits are normally passed from one generation to the next by the transfer of genetic material (DNA or RNA). Prions are proteins that aggregate in a specific and controlled manner, and that can cause inheritable traits solely by the transfer of this aggregated protein factor. Prions are infectious proteins that, at least in animals, can cause disease. The prion phenomenon is of huge scientific interest for many reasons. Aggregation is a fundamental property of proteins and all proteins do it, especially when they are damaged. The aggregation of prions however is self-propagating. Prion aggregates are able to recruit normal protein, change its shape and force it into new aggregates. Prions have been the studied intensively in recent years because of the bovine spongiform encephalopathy epidemic in cattle, and the subsequent appearance of variant Creutzfeldt-Jakob disease in the UK's human population. However, these protein-based inheritable traits are not confined to animals, and a number of prions have been found in the bakers yeast Saccharomyces cerevisiae. Understanding the basic molecular details of how the prion phenomenon works has proved difficult, at least in part because there is no detailed information on the shape and structure of prion aggregates, or on how the individual proteins change shape as they aggregate. The presence of prion traits in yeast provides an exciting scientific opportunity, as yeasts are very much easier to manipulate experimentally than animal model systems such as mice. Yeast proteins are more readily isolated and produced in the large quantities required for structural analysis. Changes in those proteins are more easily made, allowing the contribution that the different parts of the molecule make to the process of self-propagating aggregation to be understood. The research progress made is therefore faster and more cost-effective. Yeast prions are therefore an ideal model system to study the molecular basis of prion-based disorders. Ure2p is a yeast protein that normally functions to help yeast regulate how they use nutrients from their environment. However, Ure2p shows prion-like behaviour in that it can convert from its normal, active form, into inactive fibres in which the normal function is lost. In our preliminary work, we have grown Ure2p fibres and solved their 3D structure at low resolution using cryo-electron microscopy and image processing. We now wish to dramatically improve the resolution of this structure. We will also solve the structure of a smaller assembly of Ure2p which appears to be the building block from which the fibres are made. This will enable us to look at how the known structure of the soluble form fits into the structure of both the fibres and the assembly intermediate, which will in turn help us to understand the ways in which the soluble form of Ure2p must change its shape to be incorporated into a prion fibre. Our programme of experiments will therefore provide fundamental information on the molecular basis of self-propagating aggregation by prions

Aggregation is a fundamental property of polypeptide chains, and its importance has been highlighted in recent times by the discovery of prions, which in animals are associated with neurodegenerative disease. The prion phenomenon is thought to occur through the self-propagating assembly of a normal cellular protein into fibrillar aggregates. Prions are transmissible between organisms in several ways, (including oral transmission via infected material for BSE/vCJD, and mother-daughter inheritance in yeast and cattle) without transfer of genetic material. Several prions have been identified in the yeast S. cerevisiae, by their non-Mendelian mode of inheritance, and provide an experimentally accessible model system to study the molecular basis of self-propagating aggregation. Evidence shows that the [URE3] trait arises from the self-assembly of Ure2p, a soluble protein involved in the regulation of nitrogen catabolism, into an insoluble, inactive fibrils. Our initial studies show that although Ure2p fibrils are poorly ordered helices, they are less variable than other prion or amyloid specimens studied to date. We have determined an initial 3D structure, using cryo-EM of fibrils grown from full-length Ure2p in native-like conditions. We will improve the resolution of our preliminary structure using single-particle methods to deal with fibril heterogeneity and disorder. We will also solve the structure of a globular assembly intermediate of Ure2p. We will then construct a molecular model for these structures using constraints derived from specific gold labelling experiments, and by using atomic structure fitting of X-ray structures and available theoretical models of Ure2p. This will enable us to determine the locations and extent of conformational change that soluble Ure2p undergoes upon incorporation into a prion fibril, and thereby give insights into the molecular basis of self-propagating aggregation and fibrillogenesis that underlies the prion phenomenon.