Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Zilm, Kurt

Abstract

Liquid-liquid phase separation (LLPS) of macromolecules allows for a variety of cellular functions such as signal transduction and regulation of gene expression and is implicated in neurodegeneration. Cellular prion protein (PrPC) as the central player in prion and Alzheimer’s diseases has been previously discovered to undergo LLPS, while its molecular underpinnings and physiological functions remain unclear. This thesis delves into the complexity of PrPC LLPS, characterizes its biophysical properties, and initiates a connection between PrPC LLPS and its physiological functions. In Chapter 2, site-specific interactions of PrPC that drive LLPS are identified, highlighting the importance of interactions between the N-terminal and C-terminal domains. Further comparison between wild-type PrPC and a pathological mutant E200K reveals that the backbone dynamics of the N-terminal domain is influenced by interactions between N-terminal and C-terminal residues and is important for LLPS. Chapter 3 focuses on poly (4-styrenesulfonic acid-co-maleic acid) (PSCMA), a molecule that has been discovered to rescue Alzheimer’s disease-related cognitive deficits by antagonizing the interaction between PrPC and amyloid-β oligomers (Aβo). PSCMA induces reentrant LLPS of PrPC and lowers the saturation concentration of PrPC by 100-fold. A maturation process after the initial LLPS was also discovered, which leads to a β-sheet-rich structure of PrPC and is controlled by PSCMA and mutation. The final chapter examines the phase transitions of PrPC in the context of PrPC-Aβo hydrogel formation, the very first step of an Alzheimer’s disease pathway. PSCMA is shown to prevent PrPC-Aβo formation through LLPS, and the competitive nature of PSCMA is investigated by further examining the binding interface of PrPC and Aβo. The biological function of PrPC LLPS is challenging to study since the function of the protein itself is unclear. This thesis approaches this problem through mutagenesis and the use of antagonist PSCMA in addition to the fortuitous discovery of a maturation process. The instability of the intermediate state of the pathological mutant E200K before maturation, coupled with the fact that the antagonist PSCMA stabilizes this intermediate state, leads to a hypothesis that this PrPC intermediate state might be an entry point to a pathway where it can still be rescued by PSCMA or other reentrant LLPS inducers before further misfolding or aggregation. This may be key to understanding the molecular mechanisms of PrPC in neurodegenerative diseases including prion and Alzheimer’s disease.

Download

Share

COinS