Modulating conformational equilibria of prion protein and proteasome to tune proteostasis network

Many apparently unrelated pathologies termed as “protein conformational diseases” (PCDs), including Alzheimer’s (AD), Parkinson
(PD) and prion diseases, are characterized by abnormal accumulation of misfolded, toxic proteins. A hallmark of PCDs is the failure of
the proteostasis network (PN), a term which stands for the delicate equilibrium regulating protein synthesis, folding, and clearance.
Hence, the proteasome, an enzyme with specific tasks in protein degradation, is increasingly considered as an attractive target for
the development of novel approaches to the treatment of PCDs. Proteasome conformational changes induced by specific regulatory
particles (RPs) play a crucial role in driving substrate proteolysis by the catalytic core particle (CP). To be degraded, however,
proteins must first unfold and diffuse through a narrow pore at the center of the CP attributing, in turn, a key regulatory role to the
conformational changes of the substrate.
This project aims to i) unlock the details of CP/RP recognition, deepening our understanding of proteasome functioning, ii) enable
novel strategies to impair prion protein pathogenic conversion and characterize its interaction with CP, iii) develop small molecules
able to modulate such processes. To address this complex scenario, we will synergistically employ a combination of NMR, molecular
modelling, ESI/MALDI mass spectrometry, and biochemical methods. Based on the notion that distinct Prion protein (PrP)
conformations differently affect proteasome assemblies and function, we will first use PrP as an exemplary model to shed light on the complex interplay occurring between proteasome/substrate conformational equilibria. On the basis of our recent NMR studies of
PrP conformational equilibria, we will investigate the molecular basis of aberrant conformational PrP transitions mostly addressing
the characterization of very early conformational events as well as the elucidation of the unfolding mechanisms leading to the
formation of misfolded PrP species and their specific interactions with proteasome particles. Next, based on previous results
obtained in our labs and inspired by the distinct CP conformational changes induced by 19 S and PA28, we will design and synthesize
a small library of multifunctional porphyrin and polyphenol derivatives able to regulate either PrP folding dynamics and/or
proteasome function. We will perform biophysical and biochemical assays to evaluate the potential of these small molecules to i)
augment proteasome activity, ii) interfere with PrP/CP assemblies, and iii) inhibit abnormal PrP conformational transitions. Newly
synthesized compounds will be also probed for their ability to restore the PN of amyloidogenic peptides as β-amyloid and amylin,
both in tube tests and in neuronal model cells. This interdisciplinary project is expected to provide new standards of intervention in
the design of new molecular entities for the therapy of these incurable diseases.