Basic Science and Pathogenesis.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• An optimized activator peptide, NLSYYT, derived from human 19S, efficiently degrades tau protein in vitro when fused to archaeal proteasome activator PA26.
• Structure-activity relationship studies indicate that steric forces from leucine at position 5 (P5) are key for full 20S proteasome gate opening and tau degradation.

Purpose of the Study:

• To investigate the role of the P5 leucine in the NLSYYT peptide on proteasome activation and tau degradation.
• To elucidate the structural mechanisms by which P5 mutations affect 20S proteasome gate dynamics and affinity.

Main Methods:

• Proteasomal activity assays were performed using NLSYYT-derived peptides with mutations at P5.
• Fluorescence polarization and OpenSPR measured the affinity and activity of PA26-mutant complexes.
• Cryo-electron microscopy (Cryo-EM) determined structures of PA26 mutants bound to the 20S proteasome.
• In vitro and cell-based assays tracked tau and phosphorylated tau (p-tau) degradation.

Main Results:

• Mutations at the P5 leucine position significantly reduced proteasomal activity (40%-90%) and weakened affinity to the 20S proteasome.
• Cryo-EM structures revealed that P5 leucine's steric interactions with R20 of PSMA5 are essential for allosteric gate opening.
• Mutations like P5W or P5Y disrupted this allosteric network, leading to partial gate opening and reduced tau degradation efficiency.

Conclusions:

• The P5 leucine in the NLSYYT peptide is critical for 20S gate stability and function.
• Partial opening of the 20S gate due to P5 mutations hinders tau entry and degradation.
• This understanding can guide the design of proteasome activators for targeted tau degradation strategies.