Ubiquitination of tau: challenges, methods and its role in neurodegeneration

Tauopathies, among which Alzheimer’s Disease is one of the best-known representatives, are a group of neurodegenerative diseases characterised by the accumulation of intracellular protein aggregates, known as Neurofibrillary Tangles (NFTs). NFTs are primarily composed of the Microtubule-associated protein tau. The function of tau and its conversion into pathological species are critically influenced by Post-Translational Modifications (PTMs), among which the role of ubiquitination remains poorly understood. The primary objective of this doctoral thesis was to gain mechanistic insight into the effect of site-specific ubiquitination on the aggregation of tau and its liquid-liquid phase separation properties. To achieve this, we optimised two semisynthetic strategies. The first is based on disulfide chemistry, and it was optimised to install one or two ubiquitin molecules at disease-associated positions (e.g., residues 311 and 317). Our findings indicate that ubiquitination at position 317 is sufficient to completely abolish fibril formation. Furthermore, double-ubiquitinated tau lost the ability to form liquid coacervates, while monoubiquitinated samples retained this capability. The second approach is based on a novel thioether-based method, developed to produce stable conjugates (particularly, at position 353) resistant to reducing environments, thereby enabling the study of ubiquitination in complex cellular media. This thioether-based proteoform, which still fibrillates but at a reduced rate, demonstrated high stability in cellular and bacterial lysates and was successfully internalised by astrocytes. Structural and dynamic analyses (combining NMR, SAXS, IM-MS, and MD simulations) demonstrated a position and number-specific effect of ubiquitination on tau compaction, with secondary structure and local mobility largely unaffected in distant regions. Finally, we explored the interplay between ubiquitination and ERK2-mediated phosphorylation. Phosphorylated tau showed a possible protective effect against aggregation and exhibited no significant difference in liquid-liquid phase separation (LLPS) compared to unmodified tau. However, the presence of both ubiquitination and phosphorylation resulted in a reduced ability to form stable liquid droplets and a significant impact on the catalytic activity of PP2B phosphatase. In conclusion, this thesis utilised optimised semisynthetic methods to reveal that site-specific ubiquitination fundamentally modulates tau’s conformational ensemble, LLPS, and aggregation kinetics, offering critical mechanistic information to better understand the molecular basis of tauopathy pathogenesis and guiding future therapeutic research.