Elucidating the mechanism of potentiated Hsp104 NBD2 variants against proteotoxicity

Abstract

A hallmark of several neurodegenerative disorders is aberrant protein aggregation. For example, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) involve cytoplasmic aggregation of the RNA‐binding proteins TDP‐43 or FUS. Parkinson’s Disease (PD) involves the formation of toxic, cytoplasmic a‐synuclein oligomers. Resolving these protein aggregates, thus restoring them to their functional forms, is a key therapeutic goal in these diseases. Hsp104 is a hexameric AAA+ disaggregase originally found in yeast that has been found to dissolve infectious amyloid fibers (prions), suggesting that Hsp104 can be targeted to eliminate toxic aggregates in neurodegenerative diseases. However, the activity of Hsp104 against these substrates has limits, especially since TDP‐43, FUS, and a‐synuclein are not natural Hsp104 substrates. Potentiating mutants of Hsp104 can be therapeutically beneficial in that they rescue TDP‐43, FUS, and a‐synuclein aggregation and toxicity in yeast, whereas wild‐type Hsp104 is ineffective under the same conditions. Point mutations in the middle domain (MD) of Hsp104 strongly potentiate its activity but display off‐target toxicity. Nucleotide‐binding domain, NBD1 and NBD2 mutants safely potentiate Hsp104 to counter proteotoxicity in ALS‐FTD and PD without significant off‐target effects. Interestingly, several potentiating NBD1 variants have been tested but such mutations have been sparse in NBD2. NBD2 is critical for hexamerization and the site N566 is the only NBD2 site so far that can be potentiated. We suggest that NBD1 and NBD2 have different mechanisms in their ability to potentiate Hsp104 and set out to characterize the effects of N566 mutants.