Hsp100 polypeptide translocases are conserved AAA+ machines that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from S. cerevisiae solubilizes stress-induced amorphous aggregates and amyloid. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-EM structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80 Å-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore-loop-substrate interactions by two-amino acids. These changes are coupled to activation of specific ATPase sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and a remarkable structural plasticity of Hsp104-catalyzed disaggregation.

Amyloid fibrils are protein homopolymers that adopt diverse cross-β conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including:(1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases.

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...

Highlights d The dynamic assembly shows varying tunnel width, helical rise, and domain orientation d The N-terminal domain and C-terminal tail are not required for CtHsp104 hexamerization d CtHsp104 rescues TDP-43, polyGlu and a-Syn toxicity in yeast and is well tolerated d CtHsp104 confers thermotolerance to yeast suggesting collaboration with Hsp70/Hsp40

No abstract