Liquid - liquid phase separation of proteins in coacervates

Liquid-liquid phase separation (LLPS) is a phenomenon in which macromolecules partition into two distinct phases: a dilute phase and a concentrated phase. This process occurs due to various factors, including multivalent interactions and molecular crowding. LLPS plays a crucial role in cellular organization, enabling proteins, RNA, and DNA to form condensed phases known as membraneless organelles (MLOs), such as Cajal bodies, P-bodies, and stress granules. These MLOs participate in essential cellular processes, including gene expression regulation and stress response. Beyond its physiological roles, LLPS has been implicated in neurodegenerative diseases, such as Alzheimer's and Parkinson’s, due to the high concentration of proteins within liquid droplets, which can promote protein aggregation and amyloid formation. Additionally, p53, a key tumor suppressor protein, is known to undergo phase separation. p53 plays a pivotal role in cancer, activating pathways involved in apoptosis, cell cycle arrest, and DNA repair. Mutations in its core DNA-binding domain often lead to protein aggregation and gain-of-function effects that contribute to cancer progression. Studies have shown that while wild-type p53 (p53c WT) can phase separate in response to DNA damage, mutant forms do not. Some chaperones, such as Hsp90, are capable of reversing p53 aggregation and restoring its functional state. Hsp90, in particular, has been shown to protect p53c from aggregation under thermal stress. Liquid droplets formed via LLPS can be categorized into different coacervate models, including segregative and associative coacervation. In segregative coacervation, macromolecules remain in separate phases, with the droplet composition depending on macromolecule concentration. In contrast, in associative coacervation, both macromolecules coexist in the same phase. In this study, we intend to investigate coacervates formed by PDADMAC and RNA interacting with wild-type p53 core domain (p53c WT), the p53 core domain mutant R248Q (p53c R248Q), and Hsp90. The coacervates will be design to exhibit different surface charges (positive, negative, and neutral) and will be combined with the proteins. Using fluorescence microscopy, we aim to determine the localization of p53 within the coacervates and assess how p53c WT and p53c R248Q behave in the presence of Hsp90.

Finantial support: FAPESP, CNPq and CAPES