**1. Introduction**

Ubiquitination is a prevalent post-translational process that regulates essential eukaryotic cell functions such as protein homeostasis, signaling, and cellular localization [1–3]. It consists of the covalent addition of the small protein ubiquitin to the side chain of lysine residues in a target protein. The reaction is controlled by the coordinated action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3), which catalyze the formation of an isopeptide bond between the carboxyl group of the C-terminal glycine of ubiquitin and the ε-amino group of the substrate's lysine [2–5]. Ubiquitination of a ubiquitin molecule through one (or more) of its seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63) or its amino terminus produces polymers named polyubiquitin chains [2,5]. Polyubiquitin chains have different biological functions depending on the type of linkage that connects the ubiquitin units. Lys48- and Lys11-linked chains regulate proteasome-mediated protein degradation, Lys63-linked chains are involved in endocytosis, cell signaling, and cellular response to DNA damage [2,6], while the functions of the remaining polyubiquitin chain types remain less clear [7]. Each chain type exhibits

unique conformational properties that correlate with the diverse biological activities [7,8]. Prototypical polyubiquitin chains with Lys48- and Lys63- linkage display distinct structures: prevalently compact in the case of Lys48-linked chain [9] and extended in the case of Lys63-linked polyubiquitin [10]. Di fferences in polyubiquitin chain topology generate unique molecular interfaces that govern the selective recognition of cellular receptors and the fine modulation of specific cellular pathways [11].

Due to the central role of protein ubiquitination in fundamental cellular pathways, the dysfunction of the ubiquitin system is involved in the onset of many human pathologies [12]. In particular, the impairment of protein turnover mediated by the ubiquitin proteasome system (UPS) is implicated in neurodegenerative disorders, including Alzheimer's disease (AD), characterized by the accumulation of misfolded proteins [4]. The understanding of the molecular mechanisms of UPS malfunction in relation to protein aggregation has been the target of intense and recent research aimed at finding new therapeutic strategies for the treatment of these diseases. The major incidence of neurodegeneration in the elderly can be justified in part by the fact that a progressive worsening of clearance activity in aging brains may promote the accumulation of toxic and misfolded proteins within neurons [13,14]. Additionally, it is well established that ubiquitin is a key component of the intracellular deposits formed by misfolded proteins in damaged neurons [4,15] and that certain types of protein aggregates can directly inhibit or obstruct the proteasome machinery [12,16,17]. The connection between UPS and protein aggregation was extensively studied in the case of AD, a progressive brain degeneration that is still incurable and which has a major incidence worldwide.

One of the key players in AD pathogenesis is the microtubule associated protein tau, an axonal protein mainly expressed in the central and peripheral nervous system [18]. In the brain, tau protein occurs in six isoforms of di fferent length, generated by alternative splicing [19,20]. Tau protein belongs to the class of intrinsically disordered proteins. The large conformational flexibility that characterizes disordered proteins and regions allows them to carry out a variety of biological activities via distinct recognition mechanisms unfeasible for rigid proteins [21–23]. Tau protein controls the stability and assembly of microtubules, a function that is finely regulated by phosphorylation [18]. The extensively studied, 441-residue isoform (Figure 1) includes an N-terminal half that projects from the microtubule surface and a C-terminal half that promotes the assembly of microtubules. The four pseudo-repeats R1-R4 spanning residues 244-369, together with their flanking regions, constitute the microtubule binding domain (MBD) [19]. The MBD includes two hexapeptide motifs (VQIVYK and VQIINK) that are critical in promoting nucleation of tau aggregates [24]. Under pathological conditions, tau protein becomes hyperphosphorylated, detaches from the microtubules and undergoes a complex aggregation process characterized by a conformational transition to β-sheet rich structures and formation of straight and paired helical filaments (PHFs) that accumulate in neurofibrillary tangles (NFT) [19,20]. PHFs isolated from AD-brain or obtained from recombinant tau protein have been shown to be able to inhibit the proteasome [17]. The solved structure of tau filaments purified from AD-brain revealed residues 306-378 as the ordered fibril core, with the N- and C termini forming the fuzzy coat [25].

In addition to being heavily phosphorylated, tau protein isolated from AD-PHFs was found to be ubiquitinated at several lysine sites within the MBD [26–28]. Specifically, mono-ubiquitin was found to be linked to Lys254, Lys257, Lys311 and Lys317 [27], while polyubiquitin chains were found to be conjugated to Lys254, Lys311 and Lys353 [28]. PHF-tau is modified by three polyubiquitin linkages, Lys6-, Lys11-, and Lys48-, with the latter one being the most prevalent [28]. Recent cryo-EM studies revealed that, besides a proteasome-targeting role, ubiquitination of tau filaments could be structurally involved in mediating specific inter-protofilament packing [26]. Thus, it is plausible that ubiquitination severely a ffects the assembly and stability of tau filaments and the understanding of the underlying mechanism has the potential to shed new light into the molecular basis of the disease.

To study the impact of ubiquitination on the mechanism of tau protein aggregation, detailed biophysical experiments necessitate highly homogenous and uniquely modified protein samples. However, homogenous ubiquitination of lysine side chains is rarely achieved with the use of ubiquitin ligase enzymes. Indeed, we recently showed that CHIP (Carboxy terminus of Hsp70-interacting protein), an E3 ligase of tau

protein [29], ubiquitinates the protein at more than ten sites [30]. To overcome the inherent limitation of the enzymatic approach and to obtain site-specific ubiquitination of target proteins, chemists have developed a vast array of semisynthetic methods that are based on the chemical conjugation of protein precursors [31]. Chemical ubiquitination strategies based on non-native isopeptide bond formation are often easier to implement and give high yields of the product conjugates. Among these, disulfide-coupling chemistry has proven to be highly versatile, efficient and robust, and has already been applied in many studies [30–34]. As a replacement for isopeptide linkage, this semisynthetic method generates a disulfide bond between a Cys residue placed in a specific position of the target protein and the C-terminal aminoethanethiol of a ubiquitin derivative obtained by intein processing. Recently, we described the production and characterization of tau protein mono-ubiquitinated at three different positions using disulfide-directed methodology [30]. With this approach, we obtained novel insight into the diverse effects that lysine mono-ubiquitination at different sites exerts on tau protein aggregation.

Here, we introduce a method based on directed disulfide bond formation in combination with enzymatic synthesis of ubiquitin chains to obtain controlled polyubiquitination of tau protein, thus adding a further level of complexity in the study of ubiquitinated tau protein. We produced and characterized the aggregation process of tau protein species homogenously modified with di-ubiquitin molecules at position 353, one of the ubiquitinated sites found in AD-brain filaments. The versatility of the method allowed us to incorporate two types of di-ubiquitin chains (Lys48- or Lys63- chains) characterized by different structures, and to explore the impact of these prototypical ubiquitin polymers on tau protein aggregation.
