EXTRA VIEW EXTRA VIEW Prion 7:6, 457–463; November/December 2013; © 2013 Landes Bioscience The metazoan protein disaggregase and amyloid depolymerase system Hsp110, Hsp70, Hsp40, and small heat shock proteins Mariana P Torrente1 and James Shorter1,* 1Department of Biochemistry and Biophysics; 805b Stellar-Chance Laboratories; Perelman School of Medicine; University of Pennsylvania; Philadelphia, PA USA baffling aspect of metazoan pro- Introduction A teostasis is the lack of an Hsp104 ortholog that rapidly disaggregates Controlling the “quality” of proteins and reactivates misfolded polypeptides beyond translation is key in assuring cor- trapped in stress induced disordered rect cellular functioning and preserving aggregates, preamyloid oligomers, or organismal health.1 For a nascent poly- amyloid fibrils. By contrast, in bacteria, peptide chain, folding into the correct protozoa, chromista, fungi, and plants, native structure and maintaining the cor- Hsp104 orthologs are highly conserved rect native form is challenging within the and confer huge selective advantages in crowded environment of a cell.2-4 Thus, stress tolerance. Moreover, in fungi, the proteins occasionally assume defective, amyloid remodeling activity of Hsp104 nonfunctional conformations. To cir- has enabled deployment of prions for cumvent this fundamental problem of cell various beneficial modalities. Thus, a biology, nature has devised several mecha- longstanding conundrum has remained nisms to prevent or resolve protein mis- unanswered: how do metazoan cells folding in the cell. For instance, molecular renature aggregated proteins or resolve chaperones block protein aggregation and amyloid fibrils without Hsp104? Here, actively help proteins reach their native we highlight recent advances that unveil conformations.5 If a protein has been the metazoan protein-disaggregase damaged beyond repair, it can be cleared machinery, comprising Hsp110, Hsp70, by specialized degradation systems.6-8 and Hsp40, which synergize to dissolve Alas, protein misfolding and aggregation disordered aggregates, but are unable to can overcome these systems, especially rapidly solubilize stable amyloid fibrils. upon environmental stress, which can However, Hsp110, Hsp70, and Hsp40 even elicit aging and disease.9 Indeed, exploit the slow monomer exchange protein misfolding underpins several dynamics of amyloid, and can slowly devastating neurodegenerative diseases, depolymerize amyloid fibrils from their including Alzheimer disease, Parkinson ends in a manner that is stimulated by disease, Creutzfeldt-Jakob disease, and small heat shock proteins. Upregulation Huntington’s disease.10,11 of this system could have key therapeutic In the 1990s, Susan Lindquist and applications in various protein-misfold- coworkers unequivocally defined a new Keywords: protein disaggregation, ing disorders. Intriguingly, yeast Hsp104 branch in protein quality control: protein metazoa, Hsp104, Hsp110, Hsp70 can interface with metazoan Hsp110, disaggregation coupled to protein reactiva- 12-14 *Correspondence to: James Shorter; Hsp70, and Hsp40 to rapidly eliminate tion. Although it had been speculated Email: [email protected] disease associated amyloid. Thus, meta- that protein disaggregation and reactiva- 15 Submitted: 12/03/2013 zoan proteostasis is receptive to augmen- tion might occur, it had never been con- tation with exogenous disaggregases, vincingly demonstrated. Lindquist and Revised: 12/12/2013 which opens a number of therapeutic colleagues discovered a new heat shock Accepted: 12/14/2013 opportunities. protein (Hsp) in Saccharomyces cerevisiae, http://dx.doi.org/10.4161/pri.27531 www.landesbioscience.com Prion 457 Hsp104, which was found to have a key well tolerated and even neuroprotective of ‘Q-bodies’ or ‘stress foci’: punctate role in allowing cells to survive severe stress in animal systems.30,51-55 For example, cytoplasmic structures where misfolded after heat treatment (thermotolerance).14 Hsp104 rescues α-synuclein aggregation proteins are collected prior to maturation In subsequent investigations, Hsp104 was and dopaminergic neurodegeneration in a into larger inclusions.68-70 Using a series of found to solubilize large protein aggre- rat model of Parkinson disease.30 Whether Sse1 mutants,61 we determined that Sse1 gates resulting from severe heat stress and mammals boast an equivalent protein must engage both substrate and Hsp70, recover enzymatically active proteins from disaggregase has endured as a persistent promote nucleotide exchange on Hsp70, these aggregates.12 Accordingly, yeast cells unanswered question. and bind and hydrolyze ATP itself to pro- lacking Hsp104 were no longer able to mote disaggregation of disordered aggre- rapidly solubilize and reactivate proteins Hsp110, Hsp70, and Hsp40 as gates. Thus, simply providing Hsp70 from an aggregated state following ther- a novel protein disaggregase with a NEF, such as Fes1 or Snl1ΔN, in mal stress.13,16 system place of Sse1 was insufficient to promote Since then, we have learned much protein disaggregation.56 Likewise, using about the way Hsp104 functions. Hsp104 We have recently shed some light on a series of Ssa1 mutants, we determined is a ring-shaped homohexamer with this issue and have identified the mamma- that Hsp70 must engage substrate and two AAA+ nucleotide-binding domains lian disaggregase system via biochemical Hsp110, and hydrolyze ATP for protein (NBDs) per subunit that couple ATP fractionation of mammalian cytosol and disaggregation.56 Hsp40 must harbor a binding and hydrolysis to protein disag- reconstitution with pure components.56 functional J domain to promote protein gregation.17-22 Hsp104 is thought to drive The mammalian disaggregase system disaggregation, but the J domain alone protein disaggregation by threading sub- is comprised of an Hsp110 (Apg-2), an is insufficient.56 Optimal disaggregase strates through its central channel to solu- Hsp70 (Hsc70 or Hsp70), and an Hsp40 activity was achieved when the Hsp40 tion.23-26 Hsp104 disaggregates a diverse (Hdj1 or Hdj2).56 The combination of could stimulate Hsp110 and Hsp70 array of structures, ranging from stable these three proteins was found to establish ATPase activity.56 Finally, while Hsp110, amyloid to less stable disordered aggre- an active disaggregase system in the mam- Hsp70 and Hsp40 were unable to rap- gates.12,13,27-33 Hsp104 hexamers adapt malian cytosol prepared from rat liver or idly resolve amyloid conformers directly, different mechanisms of intersubunit col- sHeLa cells.56 Hsp110, Hsp70, and Hsp40 they enhanced disaggregation of Sup35 laboration to disaggregate stress-induced were able to refold proteins from large prions and α-synuclein amyloid fibrils by aggregates vs. amyloid.27,34 Hsp104 acts chemically or thermally denatured pro- Hsp104.56 alone or in concert with other molecular tein aggregates.56 Using pure proteins, we About a year later, a subsequent study chaperones to rescue aggregated polypep- established that Hsp70 and Hsp40 alone confirmed the metazoan disaggregation tides.12,20,28,32,34-36 In particular, Hsp70, are not sufficient for robust disaggregase activity exerted by Hsp110 (Apg-1, Apg- Hsp40, and small heat shock proteins activity, but must be supplemented with 2, or Hsp105), Hsp70 (Hsc70 or Hsp70), (Hsp26 and Hsp42) can synergize with Hsp110.56 Hsp110 homologs are found and Hsp40 (Hdj1 or DNAJA2) in vitro.71 Hsp104 to promote the reactivation of in all eukaryotes and contribute toward Curiously, under the in vitro conditions protein aggregates.35,37,38 Hsp104 is also thermotolerance in mammalian cells.57,58 employed the ATPase activity of Hsp110 essential for the formation and propaga- Hsp110 can serve as a nucleotide exchange was not required to promote protein dis- tion of several yeast prions; protein-based factor (NEF) for Hsp70 but also displays aggregation.71 Hsp110 appeared to con- genetic elements comprised of amyloid chaperone activity.59-64 We established that tribute primarily by acting as a nucleotide fibers that can confer advantageous self- Hsp110-Hsp70-Hsp40 disaggregase activ- exchange factor (NEF) for Hsp70. Mild perpetuating changes in protein structure ity was most effective against disordered, aggregation conditions were even estab- and function.32,39-46 amorphous aggregates.56 Indeed, Hsp110, lished where Hsc70 and the alternative Hsp104 is highly conserved in eubacte- Hsp70 and Hsp40 were unable to rap- Hsp40, DNAJA2, could disaggregate sub- ria and eukaryotes. Inexplicably, however, idly disaggregate Sup35 prions or amy- strates if provided with the Hsp70 NEFs Hsp104 has no exact homolog or ortholog loid forms of α-synuclein.56 Disaggregase Bag-1 or Snl1ΔN instead of Hsp110.71 in metazoa.47 NBD2, but not other parts of activity was conserved to the yeast homo- Under these circumstances, stimulation Hsp104, appears to be partially conserved logs.56 Thus, Sse1 (Hsp110), Ssa1 (Hsp70), of Hsp70 nucleotide exchange was suffi- in the four ER-resident AAA+ proteins: and Sis1 or Ydj1 (Hsp40) could synergize cient for disaggregation.71 torsin A, B, 2A, and 3A, as well as the to rescue proteins from large disordered Using C. elegans as a model system, mitochondrial AAA+ protein, SKD3.48-50 aggregates.56 This activity was slow in knockdown of Hsp110 in briefly heat- This deficiency of Hsp104 in animals is comparison to Hsp104- catalyzed protein shocked C. elegans resulted in persistent puzzling, as a protein that reverses protein disaggregation, which might help explain luciferase-YFP