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000103653 005__ 20240217051120.0
000103653 0247_ $$2doi$$a10.7936/6rxs-103653
000103653 037__ $$aRDM
000103653 041__ $$aeng
000103653 245__ $$aIdentifying potentiated Hsp104 variants that suppress TDP-43 toxicity using high-throughput screening
000103653 260__ $$bWashington University in St. Louis
000103653 269__ $$a2024-02-16
000103653 270__ $$mmjackrel@wustl.edu$$pMeredith Jackrel
000103653 336__ $$aComputationalNotebook
000103653 518__ $$d2018-09-04/2024-01-08$$oCreated
000103653 520__ $$aProtein misfolding underpins numerous fatal neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein-remodeling factor from yeast, solubilizes disordered aggregates and amyloid. Hsp104 can disaggregate misfolded proteins implicated in neurodegeneration, however this activity is weak, which has prompted efforts to engineer Hsp104 variants with enhanced activity. Many potentiated Hsp104 variants have been generated, however application of these variants in mammalian systems has stalled due to suboptimal substrate specificity and off-target effects of the variants. Here, we present a high-throughput approach to identify substrate-optimized enhanced Hsp104 variants that solubilize preformed α-syn and TDP-43 aggregates with diminished off-target toxicity in mammalian cells. Mechanistic studies of these new variants also reveal new insights into Hsp104 function and potentiation. We anticipate that our screening approach could be broadly applied to a range of protein engineering targets and establish that the new Hsp104 variants we have uncovered have key improvements facilitating their application in mammalian cells.
000103653 536__ $$oNational Institutes of Health$$qhttps://ror.org/01cwqze88$$rROR$$cF31GM140622
000103653 536__ $$oCenter for Science and Engineering of Living Systems (CSELS) Fellowship
000103653 536__ $$oNational Institutes of Health$$qhttps://ror.org/01cwqze88$$rROR$$cR35GM128772
000103653 536__ $$oTarget ALS Springboard Award
000103653 536__ $$oFrick Foundation for ALS Award
000103653 536__ $$oALS Association Award
000103653 540__ $$aCreative Commons Attribution Non-Commercial No-Derivatives (CC BY-NC-SA) 4.0 International$$uhttps://creativecommons.org/licenses/by-nc-nd/4.0/
000103653 650__ $$aChemical sciences
000103653 650__ $$aBiological sciences
000103653 6531_ $$aHsp104
000103653 6531_ $$aprotein misfolding
000103653 6531_ $$aprotein disaggregase
000103653 6531_ $$aTAR DNA-binding protein 43 (TDP-43)
000103653 6531_ $$anext-generation sequencing
000103653 6531_ $$adeep mutational scanning
000103653 6531_ $$aalpha-synuclein
000103653 6531_ $$afused in sarcoma (FUS)
000103653 655__ $$aText
000103653 7001_ $$aRyan, Jeremy$$1https://orcid.org/0000-0002-0410-2456$$uWashington University in St. Louis$$4https://ror.org/01yc7t268$$5ROR
000103653 7001_ $$aStaller, Max$$1https://orcid.org/0000-0001-9094-5697$$uUniversity of California, Berkeley$$4https://ror.org/01an7q238$$5ROR
000103653 7001_ $$aJackrel, Meredith$$1https://orcid.org/0000-0003-4406-9504$$uWashington University in St. Louis$$4https://ror.org/01yc7t268$$5ROR
000103653 791__ $$tRyan, J. (2024). Engineering and evolving substrate-specific Hsp104 variants (Order No. 30990916). Available from Dissertations & Theses @ Washington University in St. Louis; ProQuest Dissertations & Theses Global. (2919616219).$$eIsSupplementTo$$aDissertation$$whttps://www.proquest.com/dissertations-theses/engineering-evolving-substrate-specific-hsp104/docview/2919616219/se-2$$2URL
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000103653 974__ $$aWashington University in St. Louis
000103653 980__ $$aWashU Researcher Data