Elucidating the role of nucleophosmin in protein quality control mechanisms inside the nucleolus.

Abstract

Irreversible protein misfolding and aggregation is associated with many human diseases, including neurodegenerative diseases such as Huntington's and Parkinson's disease. Complex quality control mechanisms and response pathways serve to maintain protein homeostasis in cells. One stress response pathway involves the nucleolus, which accumulates misfolded nuclear proteins under proteotoxic stress conditions. The nucleolar protein, nucleophosmin (NPM1), located in the granular component (GC) region of the nucleolus, interacts with misfolded proteins, reducing their mobility and thus mitigating irreversible aggregation. NPM1 undergoes liquid-liquid phase separation (LLPS) with nucleolar proteins and RNA via homotypic and heterotypic interactions involving its intrinsically disordered region, a process we propose mediates the nucleolar stress response pathway. We hypothesize that misfolded proteins interact with NPM1 through the oligomerization domain and/or the intrinsically disordered region of NPM1 and form phase-separated dynamic assemblies that prevent irreversible aggregation. Under heat stress, certain “model” proteins unfold and migrate to the GC phase of the nucleolus and interact with NPM1. To investigate how the nucleolar partitioning of the model proteins changes in response to their misfolding, we performed fluorescence confocal microscopy on the NPM1-mCherry fusion protein and different model proteins fused with mEGFP in transiently transfected HEK293T cells. We imaged under normal conditions, under heat stress, and during a recovery period to investigate the reversibility of the model proteins’ association with NPM1 in the nucleolus. Next, we will perform in vitro phase separation assays to understand how model protein misfolding affects LLPS with NPM1 in a reconstituted GC phase. The proposed studies will elucidate the structural mechanisms that promote the association of misfolded proteins with NPM1. Understanding the mechanisms underlying nuclear protein stress responses may provide new directions for the development of therapeutics against neurodegenerative diseases in the future.