Engineering and Evolving Substrate-Specific Hsp104 Variants
Abstract (summary)
Protein misfolding underpins numerous fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson's disease (PD). Currently there are no effective treatments for protein-misfolding disorders. Hsp104, a conserved hexameric AAA+ protein-remodeling factor from yeast, solubilizes disordered aggregates and amyloid. Hsp104 has only limited activity against amyloid proteins that aggregate in humans, but it can be re-engineered to solubilize these disease-associated aggregates and amyloid. Numerous potentiated Hsp104 variants have been identified, and they harbor mutations to both conservative and non-conservative residues in several regions throughout the protein. While numerous mutations can potentiate Hsp104, and many details about the structural basis for Hsp104 activity have been recently delineated for the wild-type protein, the specific mechanism of potentiation has remained unclear. Furthermore, application of Hsp104 has stalled due to suboptimal properties of the variants. Here, we will present new approaches to better understand the mechanism of Hsp104 potentiation and new engineering approaches to develop enhanced variants that rescue the toxicity of proteins implicated in ALS/FTD and PD without conferring off-target effects. First, using a comprehensive scanning mutagenesis approach, we have screened a library of Hsp104 variants to define the drivers of Hsp104 potentiation via its middle domain (MD). We find that modulation of the helix 2-helix 3/4 interface of the MD potentiates Hsp104, whereas similar mutations in the helix 1-2 interface do not. Using purified proteins, we find that broad destabilization of the MD is linked to potentiation. To further empower the development of enhanced disaggregases, we have developed a high-throughput approach to engineer enhanced variants that rescue the toxicity of proteins implicated in ALS/FTD and PD without conferring off-target effects. Using this approach we isolated several new substrate-optimized potentiated Hsp104 variants that inhibit α-synuclein seeding in mammalian biosensor cells with remarkably diminished toxicity upon expression in mammalian cells. In addition, we characterized the biochemical properties of these variants, uncovering new mechanistic insight into Hsp104 potentiation. We expect our high-throughput screening approach could be broadly applied to a range of protein engineering targets. Further, we establish that the new Hsp104 variants we have uncovered have key improvements facilitating their application in mammalian cells. These variants are well-suited for use as probes to assess the effects of amyloid disaggregation in a range of model systems as well as for further exploration as possible therapeutics.
Indexing (details)
Cellular biology;
Molecular biology
0379: Cellular biology
0307: Molecular biology
