EVIDENCE OF LIQUID-LIQUID PHASE SEPARATION IN P53C INDUCED BY POLYETHYLENE GLYCOL

P53 is a tumor suppressor protein, capable of identifying cell stress and inducing
apoptosis, and is also involved in cell cycle arrest and DNA repair. It has been
shown that mutants are inactive forms and are present in half of almost all cancer
types. Liquid-liquid phase separation (LLPS) of this protein has been observed,
suggesting that regulation and aggregation conditions may occur inside these liquid
droplets. LLPS is a formation of a liquid droplet containing one or more
macromolecules and it is often called membraneless organelles. There are many
examples of LLPS inside cells, such as Cajal bodies and P-bodies. Here we present
evidence of LLPS formation in the p53 wild-type core domain and also in mutant
form (R248Q), both induced by a crowding agent (polyethylene glycol, PEG 10% and 20%). Droplet formation was observed through optical density at 600 nm in order to measure its turbidity. It has been observed that as soon as protein is added to the PEG solution, optical density starts to grow, indicating the formation of scattering objects. Once the protein concentration is high in the PEG solution, OD values reach a plateau and then start to decrease. This may indicate droplet coalescence, decreasing the number of scattering objects. In order to investigate these findings, optical microscopy and confocal microscopy are being made. We expect to observe droplets and measure their size. For lower OD values, fewer and smaller are the droplets, and for greater OD values, there will be more droplets in a bigger size. Dynamic Light scattering (DLS) will be used to identify protein aggregation in the samples. It will be useful for interaction assays with Hsp90 and Hsp101 chaperones. It is expected that the Hsp90 chaperone will protect p53c from aggregation over time and Hsp101 will disaggregate the protein. LLPS will be also induced in these systems and dyes will be introduced for further confocal measurements in order to verify the presence of both chaperone and client protein inside droplets.