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Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by misfolded conformers. Moreover, a disruptive technology of this nature would eliminate self-templating conformers that spread pathology and catalyze formation of toxic, soluble oligomers. Here, we highlight our efforts to engineer Hsp104, a protein disaggregase from yeast, to more effectively disaggregate misfolded proteins connected with PD, ALS, and FTD. Remarkably subtle modifications of Hsp104 primary sequence yielded large gains in protective activity against deleterious α-synuclein, TDP-43, FUS, and TAF15 misfolding. Unusually, in many cases loss of amino acid identity at select positions in Hsp104 rather than specific mutation conferred a robust therapeutic gain-of-function. Nevertheless, the misfolding and toxicity of EWSR1, an RNA-binding protein with a prion-like domain linked to ALS and FTD, could not be buffered by potentiated Hsp104 variants, indicating that further amelioration of disaggregase activity or sharpening of substrate specificity is warranted. We suggest that neuroprotection is achievable for diverse neurodegenerative conditions via surprisingly subtle structural modifications of existing chaperones.

It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures, including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate noncooperatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits cooperatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates, but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable "cross-β" cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and nonamyloid clients, which impose distinct mechanical demands., (Copyright © 2012 Elsevier Inc. All rights reserved.)

De novo designed peptides that self-assemble into cross-β rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-β peptide KFE 8 , compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE 8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE 8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE 8 fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE 32 -GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-β-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. Biomaterials comprised of self-assembling peptides hold great promise for the development of new vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-β rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification. [Display omitted] [ABSTRACT FROM AUTHOR]

Hsp104, a yeast protein disaggregase, can be potentiated via numerous missense mutations at disparate locations throughout the coiled‐coil middle domain (MD). Potentiated Hsp104 variants can counter the toxicity and misfolding of TDP‐43, FUS, and α‐synuclein, proteins which are implicated in neurodegenerative disorders. However, potentiated MD variants typically exhibit off‐target toxicity. Further, it has remained confounding how numerous degenerate mutations confer potentiation, hampering engineering of therapeutic Hsp104 variants. Here, we sought to comprehensively define the key drivers of Hsp104 potentiation. Using scanning mutagenesis, we iteratively studied the effects of modulation at each position in the Hsp104 MD. Screening this library to identify enhanced variants reveals that missense mutations at 26% of positions in the MD yield variants that counter FUS toxicity. Modulation of the helix 2–helix 3/4 MD interface potentiates Hsp104, whereas mutations in the analogous helix 1–2 interface do not. Surprisingly, we find that there is a higher likelihood of enhancing Hsp104 activity against human disease substrates than impairing Hsp104 native function. We find that single mutations can broadly destabilize the MD structure and lead to functional potentiation, suggesting this may be a common mechanism conferring Hsp104 potentiation. Using this approach, we have demonstrated that modulation of the MD can yield engineered variants with decreased off‐target effects. [ABSTRACT FROM AUTHOR]

Aberrant protein folding underpins many neurodegenerative diseases as well as certain myopathies and cancers. Protein misfolding can be driven by the presence of distinctive prion and prion-like regions within certain proteins. These prion and prion-like regions have also been found to drive liquid-liquid phase separation. Liquid-liquid phase separation is thought to be an important physiological process, but one that is prone to malfunction. Thus, aberrant liquid-to-solid phase transitions may drive protein aggregation and fibrillization, which could give rise to pathological inclusions. Here, we review prions and prion-like proteins, their roles in phase separation and disease, as well as potential therapeutic approaches to counter aberrant phase transitions. [ABSTRACT FROM AUTHOR]