HyT-Tau-CPP

Amino Acid Sequence

3D Structure (/structures/AF-P10636-F1-model_v6.cif)

Function

Promotes microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. The C-terminus binds axonal microtubules while the N-terminus binds neural plasma membrane components, suggesting that tau functions as a linker protein between both. Axonal polarity is predetermined by TAU/MAPT localization (in the neuronal cell) in the domain of the cell body defined by the centrosome. The short isoforms allow plasticity of the cytoskeleton whereas the longer isoforms may preferentially play a role in its stabilization.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To confirm that HyT-Tau-CPP-mediated Tau degradation was the proteasome dependent, we incubated the Tau-EGFP overexpressed cells with 150 μM HyT-Tau-CPP for 18 h. The cells were also treated with 10 μM proteasome inhibitor MG132 for 6 h before harvest. Tau levels were analyzed by western blot assay. The results showed that the proteasome inhibitors MG132 blocked HyT-Tau-CPP-mediated degradation, which indicated that HyT-Tau-CPP-mediated Tau degradation depended on the proteasome (Fig. 4A and ESI Fig. S5E–I†).

Description

The results of western blot also showed significant decline of intracellular Tau level (Fig. 3C and D; quantified in ESI Fig. S4C and D; ESI Fig. S5A–D†). The maximal degradation level was approximately 80% in a dose-dependent manner and 75% in time-course experiment (Fig. 3C and D).

Description

To evaluate the degree of HyT-Tau-CPP-mediated cytotoxicity, a 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl tetrazolium bromide (MTT) assay was applied to measure the cell survival. Wild type N2a cells were treated with different concentration of HyT-Tau-CPP for 24 h. The quantification of cell survival showed that the cell survival was about 80% when the cells were incubated with 150 μM HyT-Tau-CPP.To evaluate the degree of HyT-Tau-CPP-mediated cytotoxicity, a 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl tetrazolium bromide (MTT) assay was applied to measure the cell survival. Wild type N2a cells were treated with different concentration of HyT-Tau-CPP for 24 h. The quantification of cell survival showed that the cell survival was about 80% when the cells were incubated with 150 μM HyT-Tau-CPP.

Description

Firstly, to evaluate the binding affinity between recombinant hTau40 protein and HyT-Tau-CPP in vitro, we appended the 5(6)-carboxyfluorescein (CF) to the N-terminus of HyT-Tau-CPP and HyT-MTau-CPP to get ‘CF-HyT-Tau-CPP’ (Fig. 1B) and ‘CF HyT-MTau-CPP’ (Fig. 1E). Recombinant hTau40 protein was expressed by Escherichia coli, BL21 strain (ESI Fig. S2A†). Then the fluorescence polarization assay was applied. The fluorescence polarization results showed that CF-HyT-Tau-CPP could bind to Tau specifically (Kd =0.7655 ±0.1882mM)andthe‘HyT MTau-CPP’ didn't have the ability to bind to Tau (Fig. 2A).