The research objective of this proposal is to define the mechanistic basis of Hsp104 a prion disaggregase, which is unknown. Hsp104 is a hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which is the only cellular factor known to rapidly disassemble amyloid or prion fibrils as well as their toxic soluble oligomeric precursors. This catalytic amyloid-disaggregase activity is remarkable because cross-? amyloid is one of the most stable protein-based structures in nature. Moreover, this activity is unique to Hsp104 and is not achieved by any other (AAA+) protein, including ClpX. Even the bacterial Hsp104 homologue, ClpB, which can disaggregate environmentally denatured protein aggregates (like Hsp104), cannot disaggregate amyloids or prions. This Hsp104 activity has enabled yeast to harness multiple prions for beneficial purposes. By contrast, in humans (which curiously lack a direct Hsp104 homologue) prions, amyloids and toxic soluble oligomers cause several fatal neurodegenerative disorders, including Parkinson's disease (PD). Protein aggregation and amyloid formation also plague the purification of recombinant proteins for basic studies and therapeutic purposes. Thus, Hsp104 offers an unparalleled opportunity to: eradicate amyloid (and toxic soluble oligomers)~ understand how amyloid (and toxic soluble oligomers) can be disaggregated~ and understand how AAA+ architecture has been adapted for this modality. Hsp104 could even be specifically enhanced and developed as: (a) an agent to increase protein solubility in diverse expression systems to enable facile purification of recalcitrant proteins for structural and mechanistic studies, and valuable therapeutic proteins for patients~ and (b) a potential therapeutic agent and mechanistic probe for diverse protein-misfolding disorders. Indeed, we have established Hsp104 as the only cellular factor known to dissociate ?-synuclein (?-syn) oligomers and amyloids and rescue ?-syn-induced neurodegeneration in the substantia nigra of a rat PD model. To develop these potential Hsp104 utilities further, it is critical to understand Hsp104 mechanism, which despite intense investigation remains poorly defined. It is unknown how Hsp104 monomers collaborate within the hexamer to promote prion or protein disaggregation or how Hsp104 engages and eradicates prions. It is also unknown how conformational changes in Hsp104 hexamers facilitate function. We have established key assays and collaborations with leading experts to meet these challenges. Based on our preliminary data, we hypothesize that hexamer plasticity enables Hsp104 to adapt distinct mechanisms to dissolve diverse aggregated structures, including prions. Here, we will combine pure protein biochemistry, biophysics and yeast biology to define the mechanistic basis of Hsp104's amyloid- disaggregase activity via three specific aims: (1) Define how individual subunits of Hsp104 hexamers collaborate to enable protein disaggregation~ (2) Define how Hsp104 engages and deconstructs Sup35 and Ure2 prions~ and (3) Define conformational changes of the Hsp104 hexamer during its ATPase cycle.

Public Health Relevance

Protein aggregation is a severe problem in several fatal neurodegenerative diseases, including Parkinson's disease (PD), as well as in the production of valuable recombinant therapeutic proteins in the biopharmaceuticals sector. Our proposed studies should provide a detailed mechanistic understanding of an enzyme, Hsp104, which reverses aberrant protein aggregation. Realization of our objectives will enable potentially game-changing solutions to eradicate protein aggregation in diverse neurodegenerative diseases, including PD, and allow facile production of proteins for therapeutics as well as for critical structural studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM099836-02
Application #
8595319
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Wehrle, Janna P
Project Start
2013-01-01
Project End
2016-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
2
Fiscal Year
2014
Total Cost
$262,931
Indirect Cost
$91,931
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
McGurk, L; Mojsilovic-Petrovic, J; Van Deerlin, V M et al. (2018) Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis. Acta Neuropathol Commun 6:84
Shorter, James; Houry, Walid A (2018) Editorial: The Role of AAA+ Proteins in Protein Repair and Degradation. Front Mol Biosci 5:85
McGurk, Leeanne; Gomes, Edward; Guo, Lin et al. (2018) Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization. Mol Cell 71:703-717.e9
Boeynaems, Steven; Alberti, Simon; Fawzi, Nicolas L et al. (2018) Protein Phase Separation: A New Phase in Cell Biology. Trends Cell Biol 28:420-435
Subudhi, Ipsita; Shorter, James (2018) Ubiquilin 2: Shuttling Clients Out of Phase? Mol Cell 69:919-921
Bogaert, Elke; Boeynaems, Steven; Kato, Masato et al. (2018) Molecular Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains in Toxicity. Cell Rep 24:529-537.e4
Chuang, Edward; Hori, Acacia M; Hesketh, Christina D et al. (2018) Amyloid assembly and disassembly. J Cell Sci 131:
Gomes, Edward; Shorter, James (2018) The molecular language of membraneless organelles. J Biol Chem :
Guo, Lin; Kim, Hong Joo; Wang, Hejia et al. (2018) Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains. Cell 173:677-692.e20
Michalska, Karolina; Zhang, Kaiming; March, Zachary M et al. (2018) Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events. Structure :

Showing the most recent 10 out of 44 publications