High-throughput screening approach identifies substrate-selective Hsp104 variants that counter amyloid seeding with...

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Hsp104 (heat shock protein 104) is a yeast protein-disaggregase crucial for protein homeostasis.
• Misfolded protein aggregates, like alpha-synuclein and TDP-43, are implicated in neurodegenerative diseases.
• Existing Hsp104 variants have limitations for mammalian applications due to suboptimal properties.

Purpose of the Study:

• To develop a high-throughput screening (HTS) approach for identifying enhanced Hsp104 variants.
• To generate Hsp104 variants with improved properties for potential therapeutic applications in neurodegenerative diseases.

Main Methods:

• Coupling a live-or-die yeast-based selection system with next-generation sequencing for parallel screening.
• Quantitative analysis of Hsp104 variant performance in yeast and mammalian cell models.
• Assessing aggregate solubilization, seeding inhibition, and splicing restoration of target proteins.

Main Results:

• Identified novel Hsp104 variants with enhanced protein disaggregation capabilities.
• Demonstrated efficacy in solubilizing alpha-synuclein and TDP-43 aggregates.
• Showed inhibition of alpha-synuclein fibril seeding and restoration of TDP-43 splicing in mammalian cells.
• Observed diminished off-target toxicity and distinct changes in ATP hydrolysis in improved variants.

Conclusions:

• The developed HTS approach enables efficient identification of superior Hsp104 variants.
• These enhanced variants hold potential for therapeutic strategies against protein-misfolding neurodegenerative diseases.
• The screening methodology is broadly applicable for protein engineering and high-throughput variant analysis.