Conformational and state transitions of amyloid-forming proteins: influence of cofactors, biomolecules, and ubiquitination

In recent years, intrinsically disordered proteins (IDPs) have attracted the interest of researchers due to their prominent role in the onset and progression of several neurodegenerative diseases. IDPs are proteins lacking secondary and tertiary structure, which regulate fundamental mechanisms within the cell, however their disordered character is often associated with abnormal behavior and disease. α-synuclein and Tau are two IDPs that became infamous due to their deposition in the form of insoluble aggregates in Lewy bodies (LBs) and in Neurofibrillary tangles (NFTs) in the brain of patients affected by Parkinson’s and Alzheimer’s diseases, respectively. The latter are the most common among a larger group of devastating disorders in which irreversible aggregation of those proteins takes place. The aggregated, fibrillar state of the proteins is thought to form via a nucleation-growth mechanism, based on vitro investigations. In fact, monitoring the aggregation kinetics of aggregation-prone proteoforms can provide useful insights into the determinants of aberrant conformational transitions. The possibility to change the aggregation kinetics and redirect aggregation pathways is under intense scrutiny in the scientific community in a bid to identify disease-modifying agents, which are currently unavailable for any neurodegenerative disorder. In this work, we strived to explore the effects of diverse cofactors and chemical agents on the aggregation of α-synuclein and Tau. Specifically, we focused on i) lipids, ii) coffee and coffee derivatives, and iii) nanoparticles (NPs). The human brain is enriched in lipids and particularly fatty acids (FAs). Dysregulated lipid homeostasis leads to neurological disorders like AD and PD. Abnormal levels of long-chain polyunsaturated fatty acids (LC-PUFAs) correlate with enhanced protein aggregation in general and in particular with Tau protein aggregation. Indeed, there is the evidence of association between NFTs and lipids. For these reasons, we investigated the conformational changes of Tau4RD in the presence of arachidonic acid and cis-9-octadecenoic acid (chapter 4.1.1). The potentiality of natural compounds is having a great impact on drugs and treatments discovery; indeed, their availability and biocompatibility make them widely studied. Many studies about coffee and coffee derived phenolic compounds have proven their role in neuroprotection against oxidative-stress and neuro-inflammation thanks to their ability to cross the Blood Brain Barrier. Some of these molecules, such as phenylindanes, clorogenic acid, and other flavonoids have the ability to inhibit Tau aggregation. Starting from these notions, we investigated the influence of an Italian espresso coffee mixture and of trigonelline, theobromine, genistein, and caffeine on Tau4RD aggregation (chapter 4.1.2). NPs gained a prominent role in medicine and biotechnology thanks to the wide range of possible applications, their emerging properties, and tunable surface chemistry. Most proteins will interact with the surface of NPs: indeed, when NPs are immersed in a biological fluid, a layer of tightly bound proteins called “Hard corona” and an outer layer of weakly associated proteins called “Soft corona” are formed. Here, we used silica nanoparticles (SNPs) synthetized with Stöber methodology to study α-synuclein and Ox-α-synuclein aggregation modulation (chapter 4.1.3). Tau protein, like most IDPs, is subjected to several post-translational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination, and others. Abnormal PTMs result in changes in protein conformation, localization, and function. Phosphorylation is widely studied because Tau is found to be hyperphosphorylated in NFTs. Ubiquitination is also gaining interest because an impairment in the Ubiquitin-Proteasome system (UPS) contributes to the pathogenesis of AD and other neurodegenerative disorders. Ubiquitin (Ub) is linked to substrate proteins through a mechanism mediated by an enzymatic cascade. Moreover, ubiquitin can be attached to another ubiquitin moiety forming a poly-ubiquitin chain. Depending on the type of modification, ubiquitinated proteins are degraded or regulated. Modification of substrate residues by ubiquitin can affect conformational behavior and interactions (chapters 4.2.1 and 4.2.2). Ubiquitination is a reversible process, and several deubiquitinating (DUB) enzymes act to remove ubiquitin molecules from a ubiquitinated substrate. This mechanism allows a further regulation of protein degradation and ubiquitin recycling. Among DUBs, otubain1 (OTUB1) specifically deubiquitinates tau protein and in this work, we aimed at clarifying the Tau-OTUB1 interaction (chapter 4.2.3). Tau destined for degradation is targeted to the proteasome by the shuttle protein Ubiquilin2 (UBQLN2). There is evidence of colocalization of UBQLN2 and Tau in human cells but the direct interaction between these two proteins remains unclear. Both UBQLN2 and Tau undergo liquid-liquid phase separation (LLPS) in vivo and in vitro. LLPS is a phenomenon caused by demixing of a protein solution in two phases: a dilute and a dense phase. The dense phase, in which the protein concentration is higher, appears in the form of liquid droplets observable with microscopy. It is observed that UBQLN2 condenses in membrane-less organelles called stress-granules (SGs) when the cell is subjected to stressful conditions. SGs usually contain RNA-binding proteins with intrinsically disordered regions (IDRs). Interestingly, it was shown that Tau undergoes LLPS in the presence of polyanionic RNA in vivo and without cofactors in vitro conditions too. Tau condensation might represent the initial step of its fibrillar aggregation, due to the high concentration characteristic of the condensed state. In this work, we started to investigate the Tau-UBQLN2 interaction and the ability of these two proteins to reciprocally influence their LLPS behavior (chapter 4.2.4).