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

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.

The dynamics and physical attributes of biomolecular condensates (BMs) are closely linked with their biological roles. Condensation and dissolution in response to stress is a hallmark of stress granules, whereas the conversion from liquid-like to gel-like states of p62 bodies can result in degradation via autophagosome formation. These processes are tightly regulated through two main mechanisms: 1) via post-translational modifications or 2) by changing the BM composition, specifically of the non-scaffold components (ligands). Determining how ligands affect phase boundaries within live cells is crucial for understanding how BM formation occurs in the right place at the right time. Here we propose to design novel biomolecules that can alter the physical properties of BMs and to introduce these engineered molecules into a cell model to understand how changes in the physico-chemical properties (characterised in vitro) affect the targeted degradation of liquid droplets via autophagy in the complex cellular environment. To achieve our goals, we will: 1) create novel proteins comprising molecular adhesive peptides to drive liquid-liquid-phase separation (LLPS) and a consensus tetratricopeptide repeat protein (CTPR) to endow the droplets with functional capabilities, 2) characterise LLPS in vitro to define how ligand recruitment modulates the physico-chemical properties of the LLPS-CTPRs, 3) relate the properties of the LLPS-CTPRs to autophagosome formation and subsequent protein degradation, and lastly, 4) explore the design of LLPS-CTPRs as a therapeutic strategy to enhance substrate degradation or dissipation of natural occurring BMs. Using this systematic approach, we will establish a structure-function relationship between BMs and protein degradation, ultimately enabling us to rationally design novel biomolecules that can enhance targeted degradation of disease-related proteins.