**2. Results**

In the present work, we optimized a semisynthetic strategy based on disulfide-coupling chemistry to covalently attach a di-ubiquitin molecule to the tau protein. Ubiquitin-substrate conjugates obtained using this method are functional mimics of the native ubiquitinated counterparts, with the synthetic linkage being only one atom longer than the native isopeptide bond [32,34]. We selected the construct tau4RD, which represents a short form of the protein, spanning residues Q244-E372 plus an initial Met (Figure 1). Tau4RD has been widely used to study the aggregation behavior of tau protein as it includes the majority of the microtubule binding region and most of the residues involved in the assembly of the cross-β structure forming the core of the tau filaments [35]. To obtain site-specificity, we used a tau4RD mutant devoid of the endogenous cysteines and where a single cysteine was installed at the desired position. We chose to introduce the modification at position 353 because this site was found polyubiquitinated in PHF-tau [26,28], it is part of the fibril core found in di fferent tauopathies [26], and mono-ubiquitination at this position had an observable influence on the aggregation kinetics without hindering the formation of short fibrils [30]. Thus, it was deemed suitable for evaluating the e ffect of an extended ubiquitin chain on tau filament assembly.

**Figure 1.** Schematic representation of Tau441 and Tau4RD proteins with their domain organization. The position of residue 353, that is, the conjugation site with di-ubiquitin molecules used in this work, is highlighted in red.

The method of preparation of di-ubiquitinated tau4RD is illustrated in Figure 2. First, using linkage-specific enzymes, we assembled di-ubiquitin molecules with Lys48- and Lys63-linkage (Figures 2b and 3a,b). We used the ubiquitin mutants Lys48Arg or Lys63Arg as distal units and Ub-SH, a ubiquitin derivative bearing a C-terminal aminoethanethiol linker obtained from intein cleavage with cysteamine, as proximal unit (Figure 2a). Homogenous di-ubiquitin chains ending with a C-terminal thiol (Ub2(48/63)-SH) were then purified and allowed to react with 5,5--Dithiobis(2-nitrobenzoic acid) (DTNB) to produce asymmetric activated disulfides (Figures 2c and 4a,b). Finally, the activated disulfides were incubated with tau4RD, modified with a unique cysteine at position 353, to produce the disulfide-linked conjugates Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) (Figure 2c). The protein conjugates were obtained at purity of >95% (Figure 3c) and their identity was verified by mass spectrometry (Figure 4c,d). These samples were then used to perform aggregation experiments and their behavior was compared with that of mono-ubiquitinated Ub-tau4RD(353) and of the unconjugated cysteine-free protein (tau4RDΔC).

**Figure 2.** (**a**) Scheme of cysteamine-mediated cleavage of Ub-SH from a ubiquitin-intein fusion protein. We produced a chimeric protein where ubiquitin was cloned to the N-terminal of the GyrA intein. A ubiquitin with a C-terminal aminoethanethiol linker (Ub-SH) was obtained through a trans-thioesterification reaction between intein and cysteamine, followed by a S-to-N acyl shift. (**b**) Scheme of production of Ub2(48)-SH and Ub2(63)-SH di-ubiquitin molecules by enzymatic reaction. For the controlled assembly of K48-linked chains, the enzymes E1 and E2-25K were used. For the K63-linked chains, we used E1 and the complex Mms2/Ubc13. (**c**) Reaction of Ub2(48/63)-SH with 5,5--Dithiobis(2-nitrobenzoic acid) gave an activated asymmetric disulfide. This was then allowed to react with the unique cysteine placed in position 353 in tau4RD to obtain the disulfide-linked Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) conjugates.

**Figure 3.** SDS-PAGE showing the enzymatic reaction to obtain the Ub2(63)-SH (**a**) or Ub2(48)-SH (**b**) di-ubiquitin molecules. In (**c**), the purified Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) are shown.

**Figure 4.** Maldi TOF MS analysis of protein samples. We obtained mass values of 17,393 and 17,396 for the Ub2(48)-S-TNB (**a**) and Ub2(63)-S-TNB (**b**) adducts, respectively (expected mass 17397), where TNB stands for (2-nitro-5-thiobenzoic acid). In (**a**) and (**b**), the MS peak of the unconjugated Ub2(48/63)SH protein is also present (expected mass 17200). We obtained mass values of 30,922 and 30,884 for the Ub2(48)tau4RD(353) (**c**) and Ub2(63)tau4RD(353) (**d**), respectively (expected mass 30923).

The kinetics of filament formation of the prepared proteins was followed by monitoring changes in Thioflavin T (ThT) fluorescence (Figure 5a,b). In this assay, the fluorescence emission of ThT increases upon its specific binding to the β-sheet rich structure characteristic of tau filaments. The sigmoidal profile of the kinetic curves reflects the cooperative nature of the nucleation-dependent aggregation process. The initial flat portion of the curve, corresponding to the lag phase, is followed by a steep transition (the growth or elongation phase) and a flat terminal part (plateau phase). As shown previously [30], aggregation of tau4RDΔC in the presence of heparin is very rapid, characterized by an early transition midpoint at ~5 h, and a fast fibril growth, with an elongation time of 0.7 ± 0.2 h (Figure 5a,b, Table 1). The addition of unconjugated di-ubiquitin molecules (Ub2(48/63)) at an equimolar ratio with tau4RDΔC did not affect the aggregation curves (Figure 5a), excluding the possibility that significant tau-ubiquitin inter-molecular contacts interfered with filament formation. Moreover, in the experimental conditions used, neither Lys48- nor Lys63-linked di-ubiquitin formed fibrils by themselves

(Figure 5a), thereby allowing us to interpret the experimental data in terms of ubiquitination-induced perturbations of the aggregation of tau protein.

**Figure 5.** (**<sup>a</sup>**,**b**) Thioflavin T-based aggregation kinetics experiments. Error bars of fluorescence curves correspond to standard deviations of at least three independent experiments. In (**a**), aggregation data of tau4RDΔC are reported, in the absence or presence of equimolar Ub2(48/63). Kinetics data for the Ub2(48/63) alone are also reported. For these control experiments Ub2(48/63) were produced with a distal D77 ubiquitin mutant, to avoid insertion of free thiol groups. In (**b**), aggregation data of di-ubiquitinated forms Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) are reported in comparison with tau4RDΔC, and mono-ubiquitinated Ub-tau4RD(353). (**c**) On the right, dot Blot of Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) at different times of aggregation induced with equimolar heparin, probed with the A11 antibody. On the left, deposited proteins were stained with Ponceau. Samples of tau4RDΔC and Ub-tau4RD(353) were used as controls. (**d**) Microtubule assembly in the presence of tau protein samples was measured monitoring the absorbance at 350 nm. Error bars of absorbance curves correspond to standard deviations of three independent experiments. Ub2(48)tau4RD(353) sample in the absence of tubulin was used as control.

The sigmoidal aggregation profiles obtained with Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) indicate that di-ubiquitinated tau protein samples were capable of forming filamentous aggregates (Figure 5b). From visual inspection of the aggregation curves measured on all of the tau protein samples, we noted a significant difference in the maximum fluorescence at plateau for the mono-ubiquitinated tau protein in comparison with the other proteins. Because signal intensity is influenced by specific amyloid properties and the proteins under investigation have different structures and shapes, the reduction in ThT fluorescence observed for mono-ubiquitinated tau protein was likely due to a different affinity for ThT and not to the number of fibrils formed.

Based on a quantitative analysis of the aggregation kinetics, we found that filaments formation by di-ubiquitinated tau protein was significantly delayed compared to the unconjugated protein, as deduced by the longer lag phase and elongation time of the di-ubiquitinated proteins (Figure 5b, Table 1). Additionally, di-ubiquitination was found to specifically affect the transition midpoint and elongation time more than mono-ubiquitination. Indeed, Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) displayed a similar transition half time of ~21 h, which was significantly shifted with respect to the value determined for Ub-tau4RD(353) (t0.5 ~15 h), and all the values of modified tau protein were larger compared to that of tau4RD ΔC (t0.5 ~5 h). Likewise, the elongation time showed an analogous

trend for the investigated samples (Table 1). By comparison of the aggregation curves, it emerged that the kinetics of the two di-ubiquitinated proteins was similar.

Ub2(48)tau4RD(353) and, to a lesser extent Ub2(63)tau4RD(353), produced aggregates that reacted with A11 (Figure 5c), an antibody capable of recognizing prefibrillar oligomers of diverse proteins [36]. Thus, the ability of the tau protein component to form intermediate amyloidogenic species was maintained after modification, as shown previously for mono-ubiquitinated tau4RD [30]. The ability of both Ub2(48)tau4RD(353) and Ub2(63)tau4RD(353) to form mature fibrils was confirmed by the TEM images (Figure 6a,b), which showed the presence of well-formed twisted filaments. The morphological analysis revealed that filaments were characterized by a large width of 20 ± 2 nm and a narrow width of 15–16 nm, and a twist crossover repeat of 57-58 nm (Table 2), indicating that the overall morphology of tau4RD Δ C filaments was not heavily modified by di- or mono-ubiquitin conjugation (Figure 6a–d, Table 2).

Taken together, the obtained data clearly indicate that the incorporation of protein modifiers in the microtubule binding domain of tau protein at position 353 interferes with the aggregation mechanism but does not abrogate the formation of mature fibrils. The addition of one ubiquitin moiety to tau protein determines the inhibition of fibrils formation. The inhibitory e ffect is even stronger when a second ubiquitin moiety is attached to the proximal ubiquitin unit, as it resulted from the substantial increase of the duration of both midpoint transition and elongation time for the di-ubiquitinated species. However, despite their known structural di fferences, the topology of the investigated di-ubiquitin molecules (Lys48- or Lys63-linked) did not influence the process of fibrils formation. Thus, it appears that the inhibitory e ffect of di-ubiquitination at the 353 site is caused by the increased steric hindrance which impairs microscopic events that lead to fibrils formation.

**Figure 6.** TEM representative images of (**a**) Ub2(48)tau4RD(353), (**b**) Ub2(63)tau4RD(353), (**c**) tau4RDΔC, and (**d**) Ub-tau4RD(353), after 48 h of incubation at 37 ◦C under static condition. 30 μL of sample at a concentration of 2.5 μM were deposited. Distributions of (**e**) cross-over distances and (**f**) widths of the twisted filaments measured from TEM images of Ub2(48)tau4RD(353) (1), Ub2(63)tau4RD(353) (2) and Ub-tau4RD(353) (3) conjugates and tau4RDΔC (4). In (**e**) and (**f**), dots indicate single measurements and bars the positions of means ± SD. In (**f**), dots in magenta refer to the measures of narrow widths and in black to large widths.

After having established how di-ubiquitination affects tau fibril formation, a process associated with disease, we set out to describe its consequence on tau protein functional activity. Specifically, we investigated the impact of Lys48- and Lys63-linked di-ubiquitin on tau-mediated tubulin polymerization. The assay was performed using Ub2(48)tau4RD(353) or Ub2(63)tau4RD(353), and in the presence of tau4RDΔC as a control (Figure 5d). Microtubule (MT) polymerization was monitored by following the increase in absorbance at 350 nm. The kinetics of MT assembly in the presence of tau4RDΔC resembled previous results reported on tau4RD [37]. The data acquired in the presence of Ub2(48)tau4RD(353) or Ub2(63)tau4RD(353) indicates that the presence of the di-ubiquitin chains at position 353 of tau protein, moderately but significantly inhibits MT polymerization. Indeed, after 300 min of incubation of tubulin with the conjugates, we observed ~77% of MT formation (referred to 100% for tau4RDΔC). This effect was independent from the topology of the di-ubiquitin chain, as the observed curves were almost superimposable.


 20.7 ± 1.2

 21.1 ± 3.3

Ub2(48)-tau4RD(353)

Ub2(63)-tau4RD(353)

**Table 1.** Kinetic parameters for the aggregation of tau protein samples, determined on the basis of ThT fluorescenceassays.<sup>1</sup>

1 t0.5: midpoint of the transition; τ: elongation time constant; tlag= t0.5− 2τ.

 12.1 ± 1.1

 15.1 ± 1.4  4.3 ± 0.1

 3.0 ± 0.9

**Table 2.** Morphological properties of the twisted filaments of tau protein samples obtained from the analysis of TEM images.

