We have established that the sHsps from yeast, Hsp26 and Hsp42, exert tight control over the formation of beneficial Sup35 prions. Both sHsps exerted a strong and direct inhibitory effect on Sup35 prion formation at substoichiometric concentrations. These results were surprising because sHsps commonly bind 1 substrate per ~2–3 sHsp monomers 
. Thus, the strong inhibitory effect at substoichiometric concentrations indicates that the sHsps might inhibit a rare or transient NM conformer that is critical for prion formation. Surprisingly, our results suggested that this conformer was different for each sHsp. Hsp42 targeted molten Sup35 oligomers, whereas Hsp26 targeted the self-templating ends of newly assembled prions ().
Although little was known about Hsp42, it had been suggested to work by a mechanism similar to Hsp26 to inhibit protein aggregation 
. Surprisingly, however, Hsp42 inhibited spontaneous Sup35 prionogenesis by a distinct mechanism to Hsp26. Hsp42 specifically antagonized events in the lag phase of prion formation. Hsp42 prevented and reversed the maturation of Sup35 oligomers into prion-nucleating species (, steps 2 and 3). By contrast, Hsp26 bound to newly formed prions and inhibited their seeding activity (, step 5). These two activities synergized to inhibit de novo
Sup35 prionogenesis in vitro and in vivo. To the best of our knowledge, this is the first example of two distinct sHsps working together in a synergistic manner to prevent prion formation.
The mechanistic differences between Hsp26 and Hsp42 are likely conferred by their divergent N-terminal domains. Hsp42 has an extended N-terminal domain 
, which displays no homology to other sHsps. The extended N-terminal domain of Hsp42 might enable insertion into molten Sup35 oligomers in a way that precludes prion formation.
Hsp26 chaperone activity is usually activated at heat shock temperatures 
. Unexpectedly, we found that pretreatment of Hsp26 at high temperature reduced its ability to inhibit Sup35 prionogenesis, while simultaneously enhancing its ability to prevent aggregation of a chemically denatured substrate. Our result thus reveals a fundamental difference in how Hsp26 antagonizes the aggregation of a denatured protein (GDH) and a yeast prion (Sup35). Hsp26 conformations that are ineffective against heat-denatured substrates are effective against Sup35 prions and vice versa. This difference might reflect distinct driving forces of GDH and Sup35 aggregation. GDH aggregation likely involves inappropriately exposed hydrophobic surfaces, whereas NM fibrillization likely involves polar interactions or backbone interactions or both because polar residues outweigh hydrophobic residues by ~16 to 1. At physiological temperatures, Hsp26 may be primed to inhibit prion formation, but at elevated temperatures, Hsp26 loses this ability and switches to inhibiting the aggregation of heat-denatured proteins. This switch in Hsp26 activity likely contributes to the increased levels of [PSI+
] induction at elevated temperatures 
Both Hsp26 and Hsp42 bind to preformed Sup35 fibers, but only Hsp26 binding inhibited seeding activity. However, Hsp42 increased the ability of Hsp70 and Hsp40 (Ssa1:Sis1 or Ssa1:Ydj1) to inhibit seeded assembly, potentially by recruiting Hsp70 to fiber ends. These data help explain why overexpression of Hsp26 or Hsp42 cures cells of [PSI+].
Unexpectedly, Hsp26 or Hsp42 binding destabilized Sup35 prions. Hsp26 and Hsp42 binding reduced excimer fluorescence at intermolecular contact regions. The most marked effect was at residues N-terminal to the Head region, which appeared to be forced further apart in adjacent protomers by Hsp26 or Hsp42 binding. These data suggest that Hsp26 or Hsp42 harness binding energy to alter prion architecture. Notably, these sHsp-induced alterations facilitated the disaggregation of Sup35 prions by Hsp104.
Pretreatment of Sup35 prions with Hsp42 rendered them more susceptible to rapid disassembly by Hsp104. Curiously, Hsp26 alone inhibited Hsp104. However, Ssa1 and Sis1 alleviated this inhibition and promoted more effective prion disassembly. These findings might suggest that the mechanism of Sup35 prion disassembly by Hsp104 is different in the presence of Hsp26 versus Hsp42. Further experiments are needed to explore this possibility. Importantly, Hsp26 and Hsp42 promoted elimination of Sup35 prions by Hsp104 in vivo, as overexpression of Hsp26 or Hsp42 increased [PSI+] curing by elevated Hsp104 concentration.
Hsp26 and Hsp42 also promoted rapid Hsp104-catalyzed disassembly of α-syn fibers that are connected with PD. We further demonstrated that Hsp104 directly disassembles polyglutamine fibers that are connected with HD. Hsp26 or Hsp42 boosted this activity and disaggregation was maximal in the presence of Hsp104, an sHsp, Ssa1, and Sis1.
We have established an important dichotomy between how Hsp26 and Hsp42 collaborate with Hsp104. Hsp26 promotes the disaggregation of both amyloid and non-amyloid substrates by Hsp104 in the presence of Hsp70 and Hsp40. By contrast, Hsp42 selectively promotes the disassembly of amyloid substrates by Hsp104. Thus, Hsp42 is an amyloid-specific adaptor for Hsp104. In yeast, Hsp42 appears to preferentially localize to peripheral inclusions 
that might harbor amyloid conformers that can be solubilized by Hsp104 
We have shown that in the absence of Hsp104, the Hsp110, Hsp70, and Hsp40 disaggregase system 
can slowly depolymerize amyloid fibers. Depolymerization was a slow process that required many days to complete and appeared to occur on a timescale similar to molecular recycling within amyloid fibers 
. Thus, the proteostasis network might exploit this process to slowly eradicate amyloid by either accelerating monomer dissociation from fiber ends (i.e., increasing koff
, ) or inhibiting monomer reassociation with fiber ends (i.e., decreasing kon
, ) or both. Consistent with the possibility of inhibiting monomer reassociation (decreasing kon
, ), agents that inhibit seeded polymerization of Sup35 prions (e.g., Hsp26 or Ssa1:Sis1) slowly depolymerized them over the course of many days. The relatively low number of Hsp26 monomers per molecule of substrate required for Hsp26 disaggregation activity might indicate that Hsp26 acts selectively at fiber ends. The combination of Sse1, Ssa1, and Sis1 yielded the most effective depolymerization. Given the capability of this disaggregase system to extract and refold proteins from large denatured aggregates 
, we suggest that Hsp110, Hsp70, and Hsp40 might also accelerate monomer dissociation events (increasing koff
, ). Importantly, destabilization of NM fibers by Hsp26 or Hsp42 accelerated prion depolymerization by Hsp110, Hsp70, and Hsp40.
Intriguingly, this activity is not confined to yeast but is conserved to humans. Thus, the human sHsp, HspB5, accelerated the depolymerization of α-syn amyloid (which is connected with PD) by human Hsp110 (Apg-2), Hsp70 (Hsc70), and Hsp40 (Hdj1). Collectively, these data suggest that in metazoa, which lack an Hsp104 homologue, Hsp110, Hsp70, and Hsp40 can slowly eliminate amyloid forms by specifically exploiting the molecular recycling process (). Although amyloid depolymerization is slow and requires many days to complete, it occurs on a biologically relevant timescale, especially considering the lifespan of humans. Indeed, a massive therapeutic advance will likely come with the ability to stimulate the proteostasis network to dissolve α-syn fibers in a few days in Parkinson's patients. Our data provide proof of principle that this may indeed be possible and that pure, individual components can drive this process. Although released monomers could have a chance to reassemble into toxic oligomers, we suspect that components of the proteostasis network would prevent toxic oligomer formation. Shutting down expression of an amyloidogenic protein enables mammalian cells to slowly clear protein aggregates 
. Our findings suggest that sHsps and the Hsp110, Hsp70, and Hsp40 disaggregase system might play a crucial role in this clearance. Moreover, they suggest that potential RNA interference therapies to deplete the aggregating protein should be combined with targeted upregulation of sHsps and the Hsp110, Hsp70, and Hsp40 disaggregase system to promote clearance of existing aberrant conformers.
Another way to accelerate the disaggregation of α-syn fibers is to introduce Hsp104 
. Indeed, the combination of Hsp104 with Apg-2, Hsc70, Hdj1, and HspB5 disaggregated α-syn fibers most effectively and rapidly. Importantly, Hsp104 expression counteracts neurodegeneration associated with α-syn misfolding and polyglutamine misfolding in rodents 
. Thus, our findings suggest that boosting sHsp levels or activity might provide a powerful strategy to facilitate clearance of deleterious amyloid by either the endogenous human Hsp110, Hsp70, and Hsp40 disaggregase machinery 
or by Hsp104 in targeted therapeutic strategies