title: Isolating potentiated Hsp104 variants using yeast proteinopathy models.
abstract: Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson's disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and α-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania.
authors.forename: Meredith E
authors.initials: ME
authors.lastname: Jackrel
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania.
authors.forename: Amber
authors.initials: A
authors.lastname: Tariq
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania.
authors.forename: Keolamau
authors.initials: K
authors.lastname: Yee
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania.
authors.forename: Rachel
authors.initials: R
authors.lastname: Weitzman
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania; jshorter@mail.med.upenn.edu.
authors.forename: James
authors.initials: J
authors.lastname: Shorter
mesh_terms: D004268:DNA-Binding Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism* / Q000633:toxicity*; D010300:Parkinson Disease / Q000378:metabolism; D019151:Peptide Library; D011487:Protein Conformation; D034702:RNA-Binding Protein FUS / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D051844:alpha-Synuclein / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D019151:Peptide Library; D034702:RNA-Binding Protein FUS; D029701:Saccharomyces cerevisiae Proteins; D051844:alpha-Synuclein