Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain.

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.

Mechanistic Insights into Hsp104 Potentiation.

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 substrate-binding 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. Intriguingly, Hsp104(A503V) disaggregase activity responds to mixtures of ATP and adenosine 5'-(γ-thio)-triphosphate (a slowly hydrolyzable ATP analogue) differently from Hsp104. Indeed, an altered pattern of ATP hydrolysis and altered allosteric signaling between NBD1 and NBD2 are likely critical for potentiation. Hsp104(A503V) variants bearing inactivating Walker A or Walker B mutations in both NBDs are inoperative. Unexpectedly, however, Hsp104(A503V) retains potentiated activity upon introduction of sensor-1 mutations that reduce ATP hydrolysis at NBD1 (T317A) or NBD2 (N728A). Hsp104(T317A/A503V) and Hsp104(A503V/N728A) rescue TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), and α-synuclein toxicity in yeast. Thus, Hsp104(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.