Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

DeSantis, Morgan E.; Sweeny, Elizabeth A.; Snead, David; Leung, Eunice H.; Go, Michelle Sy; Gupta, Kushol; Wendler, Petra; Shorter, James

doi:10.1074/jbc.m113.520759

Cited by 47 publications

(93 citation statements)

References 72 publications

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“…171 This key difference likely reflects the more stringent requirement for Hsp70 for ClpB disaggregase activity compared to Hsp104. 107 In most cases, potentiated Hsp104 activity could be stimulated even further by supplementation with the Hsp70 chaperone system. 61 Thus, potentiated Hsp104 variants can still collaborate with the Hsp70 chaperone system, but do not absolutely require it for the disaggregation of disordered aggregates.…”

Section: Potentiated Hsp104 Variants Do Not Require Hsp70 and Hsp40 Fmentioning

confidence: 99%

Engineering enhanced protein disaggregases for neurodegenerative disease

Jackrel

Shorter

2015

Prion

Self Cite

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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Section: Potentiated Hsp104 Variants Do Not Require Hsp70 and Hsp40 Fmentioning

confidence: 99%

Engineering enhanced protein disaggregases for neurodegenerative disease

Jackrel

Shorter

2015

Prion

Self Cite

103

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“…Luciferase assay reagent was purchased from Promega (Madison, WI). Hsp70 (Hsp72) and Hsp40 Hdj1 were purchased from Enzo Life Sciences (Farmingdale, NY E285Q/A503V/E687Q , and Hsp104 Y257A/A503V/Y662A were purified as reported previously (52,61,69). Briefly, untagged Hsp104 was transformed into BL21-DE3 RIL cells (Agilent Technologies, Santa Clara, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Luciferase Disaggregation Assays-Luciferase reactivation was performed as described (23,61). To assemble aggregates, firefly luciferase (50 M) in luciferase refolding buffer (25 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 10 mM magnesium acetate, 10 mM DTT) with 6 M urea was incubated at 30°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

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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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“…28,29 While Hsp104 and ClpB are often assumed to function by the same mechanism, 30 we have established several key mechanistic differences between Hsp104 and ClpB. 16,26 Thus, we constructed a comprehensive series of deletion constructs to elucidate the MD requirements for Hsp104 potentiation. We …”

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confidence: 99%

Amelioration of Alpha-Synuclein in Parkinson's Disease through potentiated protein-protein interactions

Pate¹

2016

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Hsp104, a protein disaggregase from yeast, can be engineered and potentiated to counter TDP-43, FUS, or α-synuclein misfolding and toxicity implicated in neurodegenerative disease. Here, we reveal that extraordinarily disparate mutations potentiate Hsp104. Remarkably, diverse single missense mutations at 20 different positions interspersed throughout the middle domain (MD) and small domain of nucleotide-binding domain 1 (NBD1) confer a therapeutic gain of Hsp104 function. Moreover, potentiation emerges from deletion of MD helix 3 or 4 or via synergistic missense mutations in the MD distal loop and helix 4. We define the most critical aspect of Hsp104 potentiation as enhanced disaggregase activity in the absence of Hsp70 and Hsp40. We suggest that potentiation likely stems from a loss of a fragilely constrained autoinhibited state that enables precise spatiotemporal regulation of disaggregase activity. H sp104, a hexameric AAA+ protein from yeast, catalyzes the construction and deconstruction of yeast prions.

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Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

Cited by 47 publications

References 72 publications

Engineering enhanced protein disaggregases for neurodegenerative disease

Engineering enhanced protein disaggregases for neurodegenerative disease

Mechanistic Insights into Hsp104 Potentiation

Amelioration of Alpha-Synuclein in Parkinson's Disease through potentiated protein-protein interactions

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