Establishing a structure-function relationship between biomolecular condensates and protein degradation

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Our bodies contain some 100,000 proteins that enable or regulate essentially every biochemical process on which our lives depend. For these proteins to perform their normal roles, the vast majority must remain in their soluble functional states. To maintain this healthy balance, cells have evolved intricate quality control networks that identify aberrant and damaged components and destroy them. For this to occur, cells need to spatially organise their components to promote these specific reactions and processes. Phase separation, such as when oil is mixed with vinegar, is an important method of compartmentalisation used by the cell to cluster together proteins and other biomolecules for a variety of functions including to: 1) increase enzyme reactions, 2) suspend processes to alleviate cellular stress, or 3) concentrate components together for uptake by the cell's waste-disposal machinery. These droplets contain an array of different proteins and other components and, depending on their biological function, they can be very fluid in nature or they can have a more gel-like composition. In the case of some diseases, further compositional changes from a liquid-like state to irreversible solid-like structures can be harmful to the cell. How does the cell control the phase boundaries within live cells and how do they know when to form droplets in the right place at the right time? To answer these questions, it is necessary to understand the factors that control the droplet composition and characteristics. In this project we propose to design and build novel liquid-droplet forming biomolecules that can: (1) be easily manipulated to introduce site-directed changes that impact on the droplet's physical attributes (i.e. changing the fluidity of the droplet) and (2) specifically recruiting other proteins to the droplets in a controllable-manner to evaluate the impact of these binding partners. We will determine how systematic changes to the novel liquid-droplets affect formation and dissolution of the structures, both in the test-tube and inside cells using complementary experimental techniques. By incorporating a recognition site for autophagosome formation (a key process in autophagy - the cell's waste-disposal mechanism), we will monitor how the changes to droplet structure change the cell's ability to dispose of them. With this structure-function relationship established, we will design artificial phase-separating molecules that can drive the removal of any disease-causing proteins from the cell for use as therapeutics to treat disorders such as Parkinson's disease.