Semisynthetic and Enzyme‐Mediated Conjugate Preparations Illuminate the Ubiquitination‐Dependent Aggregation of Tau Protein

Abstract

In the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination‐linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four‐repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site‐specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme‐derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post‐translational modification on aberrant protein self‐assembly.

Unsaturated Fatty Acid-Induced Conformational Transitions and Aggregation of the Repeat Domain of Tau

“…Recombinant Tau 4RD , corresponding to the 244-372 sequence of human Tau-441, was expressed and purified as described previously [60]. Briefly, the protein was expressed in BL21 (DE3) cells grown in LB medium at 37 • C for 5 h, with 0.5 mM IPTG.…”

Semisynthetic Modification of Tau Protein with Di-Ubiquitin Chains for Aggregation Studies

“…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 , 31 , 32 , 33 , 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.…”

“…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.…”

Releasing the Lockdown: An Emerging Role for the Ubiquitin-Proteasome System in the Breakdown of Transient Protein Inclusions

“…When the Hsp70 capacity is limiting, such as during prolonged proteotoxic stress, cells sequester misfolded proteins in a distinct transient-QC nuclear hub (INQ). Ubiquitin conjugation may drive misfolded protein sequestration into transient-QC [23,53,54]. The finding that the amount of insoluble poly-ubiquitin conjugates is substantially increased following heat stress, suggests that the sequestered proteins retain their ubiquitin moiety [38].…”