Mechanistic Insights into Hsp104 Potentiation

Abstract

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...

FAIM Opposes Aggregation of Mutant SOD1 That Typifies Some Forms of Familial Amyotrophic Lateral Sclerosis

“…Other proteins have been shown to affect protein aggregates. Much work has been carried out with a disaggregating protein from yeast, Heat Shock Protein 104 (HSP104) by Shorter and colleagues (Torrente et al, 2016;Jackrel and Shorter, 2017), including modification to broaden and enhance its activity. But there is no vertebrate, let alone mammalian, homolog of HSP104.…”

Functional analysis of proposed substrate-binding residues of Hsp104

“…These structures provided unprecedented insights into the structure and mechanism of Hsp104. It is well-established that conserved pore-loop residues tyrosine 257 and tyrosine 662 directly contact substrate and mediate translocation [1][2][3]26]. However, in the new cryo-EM structure, additional contacts were noted between K649 and Y650 and substrate [4].…”

“…background diminishes the capacity of potentiated Hsp104 variants to counter disease-substrate toxicity [26]. Furthermore, via more subtle modulation of pore loop binding residues, the properties of potentiated Hsp104 variants can be tuned and improved in key ways.…”

Biology and Pathobiology of TDP-43 and Emergent Therapeutic Strategies

“…We have engineered potentiated variants of Hsp104, an AAA + ATPase and protein disaggregase from yeast, by introducing single missense mutations into the autoinhibitory middle domain (Jackrel and Shorter 2015). These potentiated Hsp104 variants solubilize preformed TDP-43 fibrils in vitro and suppress toxicity of TDP-43 and ALS-linked variants in yeast (Jackrel and Shorter 2014a, b, 2015; Jackrel et al 2014a, b; Sweeny et al 2015; Torrente et al 2016). Remarkably, potentiated Hsp104 variants also eliminate TDP-43 inclusions and restore nuclear TDP-43 localization in yeast, phenotypes that could be transformative if they were achieved in ALS patients (Jackrel and Shorter 2014a, 2015; Jackrel et al 2014a).…”