Abstract:Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity, and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops … Show more
“…Likewise, eukaryotic Hsp104 homologs that selectively suppress αSyn toxicity in yeast also suppress αSyn toxicity-induced neurodegeneration in C. elegans . Thus, we suggest that, like previously-defined potentiated Hsp104 variants ( Jackrel et al, 2014 ; Mack et al, 2020 ), these naturally-occurring Hsp104 variants may be able to mitigate proteotoxicity in a wide variety of circumstances, including in metazoan systems.…”
Section: Discussionmentioning
confidence: 58%
“…Thus, in these cases, disruption of interaction between the transplanted NTD:NBD1 unit and the ScMD appears to mimic potentiated ScHsp104 variants and enables suppression of αSyn toxicity. Indeed, mutations in ScHsp104 NTD and NBD1 that disrupt interactions with the MD can potentiate activity ( Mack et al, 2020 ; Sweeny et al, 2020 ; Tariq et al, 2019 ; Ye et al, 2020 ).…”
Section: Resultsmentioning
confidence: 99%
“…However, in some circumstances, these Hsp104 variants can also exhibit off-target toxicity ( Jackrel et al, 2014 ; Jackrel and Shorter, 2014 ; Jackrel et al, 2015 ). Thus, uncovering other therapeutic Hsp104s with diminished propensity for off-target effects is a key objective ( Mack et al, 2020 ; Tariq et al, 2019 ).…”
The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.
“…Likewise, eukaryotic Hsp104 homologs that selectively suppress αSyn toxicity in yeast also suppress αSyn toxicity-induced neurodegeneration in C. elegans . Thus, we suggest that, like previously-defined potentiated Hsp104 variants ( Jackrel et al, 2014 ; Mack et al, 2020 ), these naturally-occurring Hsp104 variants may be able to mitigate proteotoxicity in a wide variety of circumstances, including in metazoan systems.…”
Section: Discussionmentioning
confidence: 58%
“…Thus, in these cases, disruption of interaction between the transplanted NTD:NBD1 unit and the ScMD appears to mimic potentiated ScHsp104 variants and enables suppression of αSyn toxicity. Indeed, mutations in ScHsp104 NTD and NBD1 that disrupt interactions with the MD can potentiate activity ( Mack et al, 2020 ; Sweeny et al, 2020 ; Tariq et al, 2019 ; Ye et al, 2020 ).…”
Section: Resultsmentioning
confidence: 99%
“…However, in some circumstances, these Hsp104 variants can also exhibit off-target toxicity ( Jackrel et al, 2014 ; Jackrel and Shorter, 2014 ; Jackrel et al, 2015 ). Thus, uncovering other therapeutic Hsp104s with diminished propensity for off-target effects is a key objective ( Mack et al, 2020 ; Tariq et al, 2019 ).…”
The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.
“…Indeed, Skd3 is expressed in neurons and shifting localization of activated Skd3 to the cytoplasm could help combat cytoplasmic aggregates. Likewise, the expression of the PARL Skd3 enhanced variant in the cytoplasm of dopaminergic neurons may elicit therapeutic benefit similar to Hsp104 and engineered variants in Parkinson’s disease models ( Jackrel et al, 2014 ; Lo Bianco et al, 2008 ; Mack et al, 2020 ; March et al, 2020 ; Tariq et al, 2019 ). Future studies will further inform our understanding of how to harness Skd3 disaggregase activity therapeutically in synucleinopathies such as Parkinson’s disease and other neurodegenerative diseases connected with aberrant protein aggregation.…”
Cells have evolved specialized protein disaggregases to reverse toxic protein aggregation and restore protein functionality. In nonmetazoan eukaryotes, the AAA+ disaggregase Hsp78 resolubilizes and reactivates proteins in mitochondria. Curiously, metazoa lack Hsp78. Hence, whether metazoan mitochondria reactivate aggregated proteins is unknown. Here, we establish that a mitochondrial AAA+ protein, Skd3 (human ClpB), couples ATP hydrolysis to protein disaggregation and reactivation. The Skd3 ankyrin-repeat domain combines with conserved AAA+ elements to enable stand-alone disaggregase activity. A mitochondrial inner-membrane protease, PARL, removes an autoinhibitory peptide from Skd3 to greatly enhance disaggregase activity. Indeed, PARL-activated Skd3 solubilizes α-synuclein fibrils connected to Parkinson’s disease. Human cells lacking Skd3 exhibit reduced solubility of various mitochondrial proteins, including anti-apoptotic Hax1. Importantly, Skd3 variants linked to 3-methylglutaconic aciduria, a severe mitochondrial disorder, display diminished disaggregase activity (but not always reduced ATPase activity), which predicts disease severity. Thus, Skd3 is a potent protein disaggregase critical for human health.
“…Thus, potentiated Hsp104 variants have been generated by engineering an autoregulatory domain ( Jackrel and Shorter, 2015 ). These enhanced Hsp104 variants can rescue toxicity caused by α-synuclein, FUS and TDP-43 in yeast, Caenorhabditis elegans and mammalian cell model systems ( Jackrel et al, 2014 ; Mack et al, 2020 preprint; March et al, 2020 ; Yasuda et al, 2017 ). Fig.…”
Neurodegenerative diseases and other protein-misfolding disorders represent a longstanding biomedical challenge, and effective therapies remain largely elusive. This failure is due, in part, to the recalcitrant and diverse nature of misfolded protein conformers. Recent work has uncovered that many aggregation-prone proteins can also undergo liquid–liquid phase separation, a process by which macromolecules self-associate to form dense condensates with liquid properties that are compositionally distinct from the bulk cellular milieu. Efforts to combat diseases caused by toxic protein states focus on exploiting or enhancing the proteostasis machinery to prevent and reverse pathological protein conformations. Here, we discuss recent advances in elucidating and engineering therapeutic agents to combat the diverse aberrant protein states that underlie protein-misfolding disorders.
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