**3. Discussion**

The microtubule-associated protein tau plays a central role in the pathogenesis of AD. Besides being heavily phosphorylated [20], tau protein in PHFs from AD brains is found to be mono- or polyubiquitinated at multiple sites [26–28]. The observation that most of these sites belong to the microtubule binding region, a domain involved in the formation of the filaments core, suggests the attempt of the neurons to ge<sup>t</sup> rid of aggregated tau protein forms via the UPS. This hypothesis is supported by the fact that PHF-tau was found to be modified by Lys48- and Lys11- linked chains [28], both being signals for proteasomal degradation, and by mono-ubiquitin, recently recognized as an additional signal for proteasomal targeting [38]. These observations sugges<sup>t</sup> a possible role of ubiquitination in the formation and clearance of pathological tau protein species.

In a bid to advance our understanding of the impact of ubiquitination on tau protein aggregation and function, we recently started developing methods to attain controlled ubiquitination of tau protein samples for molecular-level investigations. In a previous work, we produced mono-ubiquitinated tau4RD samples and found that the impact of the modification on tau protein fibrillogenesis was site-dependent [30]. While ubiquitination at Lys311, located within the PHF motif of the R3 domain, essentially abolished filament formation, modification at Lys254 and Lys353 changed the aggregation kinetics but did not arrest fibrillation completely. To follow up on this study, in the present work, we set out to investigate whether a minimal polyubiquitin chain, a di-ubiquitin, could affect the aggregation behavior of tau protein.

To obtain site-specific di-ubiquitination, we combined enzyme-mediated preparation of di-ubiquitin derivatives with disulfide-directed ligation to tau4RD, expanding our approach originally developed to produce mono-ubiquitination [30]. Based on the previous findings with mono-ubiquitination, di-ubiquitin was installed at position 353, a site that was deemed suitable for evaluating the effect of an extended ubiquitin chain on filament assembly. Because polyubiquitin chains exist in a variety of topologies and structures, here we chose to investigate the prototypical Lys48- and Lys63-linked chains as being representative of polymers that preferentially adopt compact or extended conformations, respectively [7]. Lys48-linked di-ubiquitin predominantly adopts a closed conformation in which the functional hydrophobic patches of mono-ubiquitin are sequestered at the ubiquitin/ubiquitin interface [9]. In contrast, Lys63-linked di-ubiquitin chains are extended and adopt an open conformation with no direct contact between the neighboring ubiquitin subunits, thereby exposing the hydrophobic patches of mono-ubiquitin [10]. Thus, we hypothesized that different architectures of polyubiquitin chains could differently affect substrate aggregation.

The performed aggregation experiments revealed that both di-ubiquitinated tau protein conjugates were less prone to form fibrils compared to the unconjugated protein (Figure 5b), although they retained the capability to form A11-positive prefibrillar oligomeric species (Figure 5c) and twisted filaments (Figure 6). Moreover, the dimeric ubiquitin modifier increased significantly both the half-time of the transition and the fibril elongation time with respect to mono-ubiquitin, with the time constants following the order: di-ubiquitinated >> mono-ubiquitinated > unmodified (Figure 5b, Table 1). However, the topological differences between the two di-ubiquitin chains (Lys48- or Lys63-linked) tested do not differentially interfere with the process of fibrils formation.

It is established that the duration of the lag phase depends on the rates of multiple parallel microscopic processes, such as the formation of primary nuclei and their amplification through elongation and secondary nucleation processes [39]. Because variations of the former process do not affect the growth phase, while changes in elongation and secondary nucleation modify both the lag phase and the growth phase, it seems possible that di-ubiquitination of tau protein could interfere with the growth and proliferation of primary nuclei rather than with their formation. This was more evident for di-ubiquitinated, rather than mono-ubiquitinated, tau protein. Since the inhibitory effect was dependent on the length of the ubiquitin chain, the impact on tau protein aggregation is likely be a result of a combination of structural motif at the tau-ubiquitin conjugating point and of the presence of the distal ubiquitin moiety which adds steric hindrance. Yet, we do not exclude the possibility that a further elongation of the polyubiquitin chain could elicit different results, particularly if considering that longer ubiquitin chains have lower thermodynamic stability and can form fibrils themselves under specific conditions [40].

To interpret our results in the wider context of post-translational modifications of tau protein, we reviewed recent studies which explored the impact of single or multiple phosphorylation of full-length tau protein or tau4RD [37,41]. Phosphorylation of both Ser262 and Ser356 was found to significantly alter the aggregation kinetics of tau4RD [37], and single-site phosphorylation affected tau protein aggregation in a sequence-specific manner [37,41]: phosphorylation of Ser305 had a significant impact on fibril formation; however modification at Ser356 did not significantly perturb the aggregation of tau protein. Here we have shown that di-ubiquitination at 353, close to residue 356, allows the formation of fibrils, although with a reduced aggregation rate, in line with the observation that this site is excluded from the ordered core (residues 272–330) in heparin-induced tau filaments [42]. Additionally, recent cryo-EM studies show that tau filaments from brain tissues [25,26] are characterized by a long core that includes Lys353 and sugges<sup>t</sup> that bulky modifiers, such as mono- or di-ubiquitin, in that position could affect the mechanism of formation of tau protein aggregates.

Tau protein is known to play a central role in the assembly and stabilization of microtubules [18]. Therefore, we explored whether di-ubiquitination could regulate tau-mediated tubulin polymerization. Based on our observations, it emerges that the presence of either Lys48- or Lys63-linked di-ubiquitin at position 353 moderately but significantly inhibits MT polymerization. This effect was found to

be independent of the di-ubiquitin linkage topology (Figure 5d). Our findings are consistent with previous studies reporting that multiple mono-ubiquitination of tau protein reduces MT binding affinity [43], and phosphorylation at Ser356 (close to Lys353) slightly impairs MT assembly [37]. Taken together, the evidence on site-specific phosphorylated or on multi mono-ubiquitinated tau protein, and our results obtained with the di-ubiquitinated proteins indicate that post-translational modifications around position 353 do not crucially a ffect tau-assisted MT polymerization and that the functional activity of tau protein is highly dependent on the sites of modification.

In conclusion, we were able to obtain, for the first time, samples of tau protein conjugated to Lys48- or Lys63- linked di-ubiquitin at a specific site and to study the e ffect of these modifications on tau protein aggregation and function in comparison with the mono-ubiquitinated species. We demonstrated that the conjugation of tau protein to di-ubiquitin chains at the 353 position significantly delayed, but did not completely inhibit, tau protein aggregation, in analogy with mono-ubiquitination. Quantitative analysis of the aggregation kinetics revealed a more pronounced e ffect of di-ubiquitin compared to mono-ubiquitin during elongation of filaments, rather than during primary nucleation events. However, linkage topology had a minor e ffect on the measured kinetic parameters. Finally, we demonstrated that di-ubiquitination modulated the tau-mediated microtubule assembly, thus highlighting the potential role of ubiquitination in the regulation of tau protein function. For a more comprehensive overview, further e fforts will have to be directed to produce tau protein modified with longer polyubiquitin chains linked in di fferent positions.

We believe that our findings also provide crucial information in light of recent structural studies on tau filaments from degenerated brain tissues. Ubiquitin is proposed to play a structural role by mediating specific inter-protofilament packing and promoting the formation of fibril subtypes, specific of the di fferent tauopathies [26]. Moreover, future investigations are required to elucidate the synergic effects played by di fferent post-translational modifications, such as ubiquitination, phosphorylation and acetylation, in the transition of tau protein to toxic species, with the aim to understand their implications in the di fferent pathologies.
