Engineering enhanced protein disaggregases for neurodegenerative disease

Abstract

ABSTRACT. 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 a-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.

Mechanistic Insights into Hsp104 Potentiation

“…6, A, C, and E). Previously, we had established that an important property of potentiated Hsp104 variants bearing mutations in the MD was increased disaggregase activity against disordered aggregates in the absence of Hsp70 and Hsp40 (62,63,(65)(66)(67). However, our findings with Hsp104 N728A (Figs.…”

“…By contrast, Hsp104 A503V strongly suppressed ␣-syn toxicity (Fig. 3A) (35,62,63,66). This rescue was severely impaired by the Y257A mutation and ablated by the Y662A or Y257A/Y662A mutations (Fig.…”

“…Hsp104 has limited disaggregase activity against TDP-43 and FUS fibrils (62,63). Recently, we have engineered potentiated Hsp104 variants that mitigate TDP-43, FUS, and ␣-syn misfolding (62)(63)(64)(65)(66)(67). Missense mutations at disparate but specific positions in the MD or the small domain of NBD1 (immediately C-terminal to the MD) resulted in potentiated Hsp104 variants (62,67).…”

“…Interestingly, two potentiated Hsp104 variants, Hsp104 A503S and Hsp104 Y257F/A503V/Y662F , rescue neurodegeneration in the metazoan nervous system and hold promise as possible treatments for neurodegenerative disease (62,66).…”

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

“…However, it is unclear how the cell limits the aggregation propensity of these domains and if therapies targeting these domains could be developed. Efforts to enhance the ability of cells to disassemble inclusions using disaggregase chaperones show promise (Jackrel & Shorter, 2015; March et al , 2016). We thought post‐translational modifications would be an alternative and potentially more pharmacologically tractable strategy to disrupt assembly directly at its source.…”