Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor

Abstract

Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the speciesspecific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.ClpB | DnaK | Hsp100 | molecular chaperone

A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast

“…Although Hsp70 chaperones are well known mediators of chaperone-assisted degradation (3,7), it is difficult to fully distinguish a direct role for these chaperones in mediating Sdj1-L169P degradation from the likely increased tendency of the protein to aggregate in the absence of a major cytosolic Hsp70 chaperone. Nonetheless, Hsp70-type chaperones have previously been shown to be vital for Hsp104 function (57,58), and were also shown to associate with translating ribosomes (59), suggesting that Ssa1 and Ssa2 may potentially be involved in both Hsp104-and Ltn1-mediated pathways. Ssa1 and Ssa2 are 95% identical proteins and it is therefore surprising that Ssa1 is not able to compensate for the loss of Ssa2.…”

Engineering enhanced protein disaggregases for neurodegenerative disease

“…67,81 We focused our libraries on the coiled-coil MD, which is comprised of 4 a-helices and facilitates optimal ATPase activity, communication between NBD1 and NBD2, intrinsic disaggregase activity, and interactions with Hsp70 during disordered aggregate dissolution. 67,82,[151][152][153][154] Importantly, the MD is less conserved than the 2 NBDs, indicating that it can withstand various missense mutations without eliminating disaggregase functionality. 67 Indeed, the MD can even tolerate large protein insertions (e.g., insertion of lysozyme between Asn467 and Glu468 in MD helix 2) or helix replacements and yet still maintain Hsp104 disaggregase activity.…”

“…Potentiating mutations likely destabilize autoinhibitory interactions that dampen Hsp104 activity or induce structural rearrangements that mimic or enable allosteric activation perhaps akin to the effect of Hsp70 binding the Hsp104 MD. 154 It remains unclear how diverse conservative and nonconservative mutations can result in this phenotype. Mutation of specific residues might subtly perturb hexamer structure, possibly promoting enhanced flexibility, altered channel properties, and stable population of the potentiated state.…”

“…It also suggests that druglike small molecules that bind to the correct region of the MD might also enhance Hsp104 activity. 154 Moreover, post-translational modification of Hsp104 in specific regions of the MD could act as a switch to elicit potentiated activity in a reversible manner. Our findings suggest that the regulatory constraints placed on Hsp104 are simply too tight to counter TDP-43, FUS, and a-syn misfolding and toxicity under certain conditions.…”

Amelioration of Alpha-Synuclein in Parkinson's Disease through potentiated protein-protein interactions

“…Hsp104 appears to have evolved to exist in a naturally and fragilely constrained autoinhibited state, which likely enables precise spatiotemporal regulation of disaggregase activity. Thus, disaggregase activity would only be elicited when or where it is needed, perhaps by the Hsp70 chaperone system 37 or by natural prion substrates. 1, 16 It is curious that Hsp104 has not acquired secondary mutations to make accessing the potentiated state improbable.…”