Amyloid fibers are self-perpetuating protein aggregates. They self-replicate their specific ‘cross-β’ conformation at their growing ends, by converting other copies of the same protein to the ‘cross-β’ amyloid form. When amyloid fibers grow and divide with high efficiency they can be infectious, and are then termed prions. Cells have evolved a sophisticated machinery to alleviate such aberrant protein aggregation. For example, protein-remodeling factors resolve protein aggregates, molecular chaperones prevent protein aggregation, osmolytes act as chemical chaperones, and degradation systems eliminate misfolded proteins.
amyloid fiber
Cross-β conformation of an amyloid fiber

Nonetheless, these safeguards can be breached, especially as organisms age, and the consequences are often fatal. Prion and amyloid formation are associated with some of the most devastating neurodegenerative diseases confronting humankind, including Alzheimer’s disease, Parkinson's disease, and variant Creutzfeldt-Jakob disease. Yet, surprisingly, it is becoming increasingly clear that prions and amyloids are not always a problem. In fact, several have been harnessed during evolution for adaptive purposes and feature in some of the most revolutionary new concepts in biology and evolution, including protein-based genetic elements, long-term memory formation, melanosome biogenesis, evolutionary capacitance and the revelation of cryptic genetic variation. We employ biochemistry and genetics to understand the enigmatic mechanistic interfaces that exist between protein-remodeling factors, molecular chaperones, small molecules and amyloid/prion fibers or other misfolded species, and how these interfaces can be manipulated to divert pathogenic and promote beneficial phenotypic trajectories. Specifically, we are taking four broad approaches:

Hsp104
Hsp104
click to videos of Hsp104

1) Defining the mechanisms of Hsp104 function. Our major focus concerns Hsp104, a protein-remodeling factor of the AAA+ superfamily from yeast, which disaggregates denatured proteins and returns them to normal function. Hsp104 is also essential for the formation and inheritance of several yeast prions; protein-based genetic elements comprised of amyloid fibers that self-perpetuate alterations in protein form and function. Hsp104 can both construct and deconstruct self-replicating amyloid conformers of Sup35, which comprise the yeast prion [PSI+], and Ure2, which comprise the yeast prion [URE3]. We strive to understand the mechanistic basis of how Hsp104 structure enables these disaggregation activities and other prion-regulatory functions.


Hsp104 is often assisted by a supporting cast of molecular chaperones to rescue aggregated polypeptides. Most notably, Hsp70, Hsp40 and small heat shock proteins synergize with Hsp104 to promote the reactivation of protein aggregates. We wish to understand how these molecular chaperones achieve these synergistic activities. [top]

 


Sup35 fibers + Hsp104, t = 0


Sup35 fibers + Hsp104, t = 20 minutes

 

2) Applying Hsp104 to disease-associated amyloidogenesis. Inexplicably, Hsp104 has no known homologue in metazoa. Indeed, whether mammals possess an analogous protein disaggregase (AAA+ protein or otherwise) remains an important open question. This is vexing, for it would seem that a protein that reverses protein aggregation and restores protein function, would be critical in our fight against several diseases caused by aberrant protein aggregation. Hence, we engineer, evolve and apply Hsp104 to metazoan systems to antagonize and reverse the proteotoxic aggregation pathways that are intimately connected with Parkinson’s, Alzheimer’s and Huntington’s disease. We are also keen to identify whether there is a metazoan AAA+ protein that can perform a similar function to Hsp104. [top]

substantia nigraHsp104 protects dopaminergic neurons (red) against alpha-synuclein toxicity in the substantia nigra of the rat brain.

 

3) Defining how small molecules disrupt amyloid structure. We study a small molecule, 4,5-dianilinophthalimide, which dissolves Aβ42 fibers (that occur in Alzheimer's disease) and eliminates their neurotoxicity, and also disrupts prion structure and function. We are interested in defining the mechanisms by which this small molecule disrupts amyloid structure. Further, we seek to elucidate synergies between small molecules and protein-remodeling factors that may accelerate the disruption of specific amyloid oligomers and fibers. [top]

DAPH
3D structure of 4,5-dianilinophthalimide

 

4) hnRNP misfolding. Finally, we are investigating the mechanisms by which certain heterogeneous nuclear ribonucleoproteins (hnRNPs) misfold and aggregate in various neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitin positive inclusions (FTLD-U). We are also investigating methods to prevent or reverse these specific hnRNP misfolding events. [top]

 

 

 

Department of Biochemistry & Biophysics
Penn Medicine
University of Pennsylvania