Potentiated variants of Hsp104, a protein disaggregase from yeast, can dissolve protein aggregates connected to neurodegenerative diseases such as Parkinson disease and amyotrophic lateral sclerosis. However, the mechanisms underlying Hsp104 potentiation remain incompletely defined. Here, we establish that 2-3 subunits of the Hsp104 hexamer must bear an A503V potentiating mutation to elicit enhanced disaggregase activity in the absence of Hsp70. We also define the ATPase and substratebinding modalities needed for potentiated Hsp104 A503V activity in vitro and in vivo. Hsp104 A503V disaggregase activity is strongly inhibited by the Y257A mutation that disrupts substrate binding to the nucleotide-binding domain 1 (NBD1) pore loop and is abolished by the Y662A mutation that disrupts substrate binding to the NBD2 pore loop. A503V displays a more robust activity that is unperturbed by sensor-1 mutations that greatly reduce Hsp104 activity in vivo. Indeed, ATPase activity at NBD1 or NBD2 is sufficient for Hsp104 potentiation. Our findings will empower design of ameliorated therapeutic disaggregases for various neurodegenerative diseases.Protein misfolding and aggregation are associated with a wide variety of diseases, ranging from type II diabetes (1, 2) to neurodegenerative diseases, such as fatal familial insomnia (3, 4), Parkinson disease, and amyotrophic lateral sclerosis (ALS) (5-7). In Parkinson disease patients, ␣-synuclein (␣-syn) 6 forms toxic soluble oligomers as well as amyloid structures that accumulate in Lewy bodies and contribute to the death of dopaminergic neurons (8 -12). Similarly, toxic soluble oligomers and cytoplasmic inclusions of TDP-43 or FUS are associated with ALS and frontotemporal dementia (13-20). These misfolded protein conformers are recalcitrant and represent a colossal roadblock in the treatment of these diseases.Hsp104 is a 102-kDa AAAϩ ATPase (21) from Saccharomyces cerevisiae capable of dissolving disordered protein aggregates as well as dismantling amyloid fibrils and toxic soluble oligomers (22-33). It assembles into a homohexameric barrel structure with a central channel (34 -39). Hsp104 processes protein aggregates by directly translocating substrates either partially or completely through this channel (35, 36, 40 -46). Hsp104 encompasses an N-terminal domain, two nucleotidebinding domains (NBD1 and NBD2), a coiled-coil middle domain (MD), and a C-terminal domain important for oligomerization (Fig. 1A) (47). Both NBDs contain Walker A and Walker B motifs that are critical for nucleotide binding and hydrolysis, respectively (48). ATP hydrolysis takes place primarily at NBD1, whereas NBD2 has a nucleotide-dependent oligomerization function (29, 34, 49 -52).Remarkably, Hsp104 can remodel amyloid substrates alone, without the aid of any other chaperones (22,24,26,31,33,45,(53)(54)(55)(56). However, to disaggregate amorphous protein aggregates, Hsp104 usually needs to collaborate with the Hsp110, Hsp70, and Hsp40 chaperone system (23,26,30,32,57). Moreover, small heat shock proteins...

Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.

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