title: Mechanistic Insights into Hsp104 Potentiation.
abstract: Potentiated variants of Hsp104, a protein disaggregase from yeast, can dissolve protein aggregates connected to neurodegenerative diseases such as Parkinson disease and amyotrophic lateral sclerosis. However, the mechanisms underlying Hsp104 potentiation remain incompletely defined. Here, we establish that 2-3 subunits of the Hsp104 hexamer must bear an A503V potentiating mutation to elicit enhanced disaggregase activity in the absence of Hsp70. We also define the ATPase and substrate-binding modalities needed for potentiated Hsp104(A503V) activity in vitro and in vivo. Hsp104(A503V) disaggregase activity is strongly inhibited by the Y257A mutation that disrupts substrate binding to the nucleotide-binding domain 1 (NBD1) pore loop and is abolished by the Y662A mutation that disrupts substrate binding to the NBD2 pore loop. Intriguingly, Hsp104(A503V) disaggregase activity responds to mixtures of ATP and adenosine 5'-(γ-thio)-triphosphate (a slowly hydrolyzable ATP analogue) differently from Hsp104. Indeed, an altered pattern of ATP hydrolysis and altered allosteric signaling between NBD1 and NBD2 are likely critical for potentiation. Hsp104(A503V) variants bearing inactivating Walker A or Walker B mutations in both NBDs are inoperative. Unexpectedly, however, Hsp104(A503V) retains potentiated activity upon introduction of sensor-1 mutations that reduce ATP hydrolysis at NBD1 (T317A) or NBD2 (N728A). Hsp104(T317A/A503V) and Hsp104(A503V/N728A) rescue TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), and α-synuclein toxicity in yeast. Thus, Hsp104(A503V) displays a more robust activity that is unperturbed by sensor-1 mutations that greatly reduce Hsp104 activity in vivo. Indeed, ATPase activity at NBD1 or NBD2 is sufficient for Hsp104 potentiation. Our findings will empower design of ameliorated therapeutic disaggregases for various neurodegenerative diseases.
authors.affiliation: From the Department of Biochemistry and Biophysics and.
authors.forename: Mariana P
authors.initials: MP
authors.lastname: Torrente
authors.affiliation: From the Department of Biochemistry and Biophysics and the Pharmacology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Edward
authors.initials: E
authors.lastname: Chuang
authors.affiliation: From the Department of Biochemistry and Biophysics and.
authors.forename: Megan M
authors.initials: MM
authors.lastname: Noll
authors.affiliation: From the Department of Biochemistry and Biophysics and.
authors.forename: Meredith E
authors.initials: ME
authors.lastname: Jackrel
authors.affiliation: From the Department of Biochemistry and Biophysics and.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Go
authors.affiliation: From the Department of Biochemistry and Biophysics and the Pharmacology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 jshorter@mail.med.upenn.edu.
authors.forename: James
authors.initials: J
authors.lastname: Shorter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D017510:Protein Folding; D034702:RNA-Binding Protein FUS / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism
chemical_list: D006360:Heat-Shock Proteins; D034702:RNA-Binding Protein FUS; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D051844:alpha-Synuclein; C070972:HsP104 protein, S cerevisiae; D000255:Adenosine Triphosphate
keywords: TAR DNA-binding protein 43 (TDP-43) (TARDBP); alpha-synuclein; fused in sarcoma (FUS); hsp104; protein disaggregase; protein engineering; protein misfolding