Prions are a specific type of protein aggregate, consisting of only one protein, and possessing the interesting characteristic of being “infectious.” Proteins in the aggregate (in an insoluble form) have a different conformation than the same protein outside of the aggregate (in a soluble form), but if the former encounter the latter, the latter will “misfold” and join the aggregate. Though prions were first recognized in association with infectious diseases in mammals, prion-like proteins have been found in many organisms, with examples in yeast like Sup35 being particularly well studied. These prion-like proteins can form aggregates that share most of the same physical properties as prions in mammals. Importantly, however, the presence of the aggregated form of these proteins does not seem to have a significant negative effect on a cell. This suggests that many prion-like proteins actually serve some important function. These proteins are thus deemed functional prion proteins. The proteins that form both disease prions and functional prions have a specific region in their amino acid sequence referred to as the prion domain. If one deletes this sequence, the proteins lose their ability to form prions. If one adds this sequence to a typical protein, then that protein will gain the ability to form prion-like aggregates; in essence, it becomes a prion protein. Many of these proteins decrease in function when they aggregate. For instance, Sup35 performs its function much less efficiently when aggregated. However, there is at least one functional prion that sees an increase of function upon aggregation. This protein is CPEB, the homologs of which are found in the neurons of several animals. CPEB tags certain mRNAs for translation, but it can only do this when in its aggregated form.
It has been theorized, as well as demonstrated, that fusing the prion domain to different proteins can reduce their function when aggregates are present. However, it would be interesting to cause an increase in function upon aggregation. This is the basis for the project this year. In this project, we will fuse the prion domain of Sup35 to two halves of a fluorescent protein. If these halves come close together in the cell, they will combine and fluoresce. By inducing Sup35 aggregation in yeast, we hope to get our two fluorescent protein fragment-prion domain fusions to join the same aggregate. In the aggregate, the two fusion proteins should be brought in close proximity, giving the fluorescent protein fragments the chance to come together. There is evidence that proteins can join an aggregate while keeping their normal function, and that different proteins can be brought together in the same aggregate. Bringing proteins close in this way is, in theory, sufficient to increase the interaction between them. If we attach the same prion domain to two proteins that normally interact, there is a good chance that we can increase their interaction by inducing aggregation. A second possible way to check for the viability of protein-protein interaction in the prion is to use a technique called FRET. Basically this technique is a way to check if two fluorescent proteins are in close proximity. We would attach prion domains to two complete fluorescent proteins and get them both to join the aggregate. To give a simplified explanation, depending on the fluorescence signal we get from exciting one of the protein, we should be able to tell if it’s in close proximity to the other. As mentioned earlier, proximity of two proteins is usually sufficient to increase interaction.