The Hsp104 N-Terminal Domain Enables Disaggregase Plasticity and Potentiation

Sweeny, Elizabeth A.; Jackrel, Meredith E.; Go, Michelle Sy; Sochor, Matthew A.; Razzo, Beatrice M.; DeSantis, Morgan E.; Gupta, Kushol; Shorter, James

doi:10.1016/j.molcel.2014.12.021

Cited by 93 publications

(195 citation statements)

References 24 publications

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“…Specifically, we found that Hsp104 mutants that did not trim the foci, such as the Hsp104(T160M) mutant, did not cure [PSI ϩ ] when overexpressed (14). Consistent with these results, in vitro studies of Shorter and co-workers (43) showed that low concentrations of Hsp104 caused fragmentation of the amyloid Sup35 fibers, which in turn generated new prion seeds. In contrast, high concentrations of Hsp104 caused dissolution of the fibers into noninfectious aggregates and soluble protein (15,43).…”

Section: Discussionsupporting

confidence: 77%

“…Consistent with these results, in vitro studies of Shorter and co-workers (43) showed that low concentrations of Hsp104 caused fragmentation of the amyloid Sup35 fibers, which in turn generated new prion seeds. In contrast, high concentrations of Hsp104 caused dissolution of the fibers into noninfectious aggregates and soluble protein (15,43). Interestingly, unlike full-length Hsp104, a high concentration of an N-terminal Hsp104 deletion mutant, a mutant that does not cure [PSI ϩ ] when overexpressed (21), caused only fragmentation and not dissolution of the Sup35 fibers.…”

Section: Discussionsupporting

confidence: 62%

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Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs

Zhao

Rodríguez

Silberman

et al. 2017

Journal of Biological Chemistry

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The molecular chaperone, Hsp104, is a hexameric protein belonging to the triple-A ATPase family (1). Along with its roles in protein folding and conferring thermotolerance, this chaperone is essential for propagation of prions in budding yeast. Prions are infectious proteins that occur in two conformations, a properly folded non-infectious conformation and a misfolded infectious amyloid conformation (2). Yeast has more than a dozen prions, which pass from mother to daughter cells as prion seeds. The steady-state number of prion seeds is maintained by Hsp104, which severs the seeds; this severing reaction is dependent on the ATPase activity of Hsp104 and involves the presentation of the prion to Hsp104 by the molecular chaperones, Sis1 and Ssa1/Ssa2, followed by threading of the prions through the Hsp104 channel (3). Yeast prions are cured by inhibiting the ATPase activity of Hsp104; this inhibition can be caused by either overexpressing a dominant-negative Hsp104 mutant or by inactivating Hsp104 with guanidine. Curing is then caused by absence of prion seed severing, in combination with dilution of the seeds by cell division (4 -6).Surprisingly A red/white colony assay is the standard method of detecting these two conformations. In this assay, yeast having nonsense mutations in the adenine synthesis pathway are plated on limiting adenine medium. Another method of detecting these two conformations is by fluorescence imaging using GFP-labeled Sup35. Using these methods, we previously showed that the curing of [PSI ϩ ] by overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) requires a new activity of Hsp104 called trimming (13). Like severing, trimming reduces the size of the prion seeds. However, unlike severing, trimming, which may occur through removal of Sup35 molecules from the ends of the prion fibers, is not accompanied by an increase in seed number. The dissolution of the prion seeds caused by overexpression of Sc-Hsp104 was shown to require the trimming activity of Hsp104. Mutants of Hsp104 that cannot trim also cannot cure [PSI ϩ ] prion by overexpression (14). For example, overexpression of the T160M point mutant of Sc-Hsp104 that lacks trimming activity but has severing activity comparable with wildtype Sc-Hsp104 does not cure [PSI ϩ ]. Consistent with our in vivo observations, in vitro evidence for dissolution of the prion seeds by Hsp104 has been obtained by Shorter and Lindquist (15). Other chaperones and cochaperones such as Hsp70, Sti1,

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Section: Discussionsupporting

confidence: 77%

Section: Discussionsupporting

confidence: 62%

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs

Zhao

Rodríguez

Silberman

et al. 2017

Journal of Biological Chemistry

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“…Interestingly, we recently discovered that the N-terminal domain of Hsp104 (residues 1-157) is essential for the potentiated activity of Hsp104 A503V and Hsp104 A503S . 156 Typically, these potentiated Hsp104 variants reduced aggregation and toxicity of TDP-43, FUS, and a-syn in yeast without reducing their expression level or inducing a heat shock response. 61 The expression of potentiated Hsp104 variants was also similar or lower to the expression of WT Hsp104, which failed to rescue TDP-43, FUS, and a-syn aggregation or toxicity.…”

Section: 2298mentioning

confidence: 99%

Engineering enhanced protein disaggregases for neurodegenerative disease

Jackrel

Shorter

2015

Prion

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103

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ABSTRACT. Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by misfolded conformers. Moreover, a disruptive technology of this nature would eliminate self-templating conformers that spread pathology and catalyze formation of toxic, soluble oligomers. Here, we highlight our efforts to engineer Hsp104, a protein disaggregase from yeast, to more effectively disaggregate misfolded proteins connected with PD, ALS, and FTD. Remarkably subtle modifications of Hsp104 primary sequence yielded large gains in protective activity against deleterious a-synuclein, TDP-43, FUS, and TAF15 misfolding. Unusually, in many cases loss of amino acid identity at select positions in Hsp104 rather than specific mutation conferred a robust therapeutic gain-of-function. Nevertheless, the misfolding and toxicity of EWSR1, an RNA-binding protein with a prion-like domain linked to ALS and FTD, could not be buffered by potentiated Hsp104 variants, indicating that further amelioration of disaggregase activity or sharpening of substrate specificity is warranted. We suggest that neuroprotection is achievable for diverse neurodegenerative conditions via surprisingly subtle structural modifications of existing chaperones.

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“…Moreover, small heat shock proteins such as Hsp26 can enhance disaggregase activity further (30,(57)(58)(59). In vitro, mixtures of ATP and ATP␥S (a slowly hydrolyzable ATP analogue) enable Hsp104 to dissolve amorphous aggregates in the absence of Hsp70 and Hsp40 (26,35,60,61).Wild-type (WT) Hsp104 can resolve ␣-syn oligomers and fibrils, but very high Hsp104 concentrations are required (26,31,32 …”

mentioning

confidence: 99%

“…The N-terminal domain of Hsp104 is critical for Hsp104 potentiation, as is motif 1 (helix 1 and a portion of helix 2) of the MD (35,47,67). Thus, deletion of these large regions precludes potentiation (35,67).…”

mentioning

confidence: 99%

Mechanistic Insights into Hsp104 Potentiation

Torrente¹,

Chuang

Noll³

et al. 2016

Journal of Biological Chemistry

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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...

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The Hsp104 N-Terminal Domain Enables Disaggregase Plasticity and Potentiation

Cited by 93 publications

References 24 publications

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs

Engineering enhanced protein disaggregases for neurodegenerative disease

Mechanistic Insights into Hsp104 Potentiation

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