Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain

Tariq, Amber; Lin, Jia Bei; Noll, Megan M.; Torrente, Mariana P.; Mack, Korrie L.; Murillo, Oscar Hernandez; Jackrel, Meredith E.; Shorter, James

doi:10.1093/femsyr/foy042

Cited by 40 publications

(57 citation statements)

References 143 publications

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“…For example, in ALS, protein condensates formed by FUS or hnRNPA1 can transit to a more solid hydrogel or aggregate phase 8,9,19 . Initially this process can be reversed by ATP-dependent chaperones or disaggregases 67 , but the increasing spatial order of these protein assemblies then promotes a transition to a solid-like fibrous aggregate, that may be toxic for neurons and so be a driver of ALS neurodegeneration 14 . We therefore tested the impact of down regulation of RNA helicases on cell viability as an approach to defining whether RNA helicase sequestration/disruption might underpin ataxin-1[85Q]-driven cell toxicity.…”

Section: Down Regulation Of Dead/h-box Rna Helicases Decreases Ataxinmentioning

confidence: 99%

Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics

Zhang

Hinde

Schneider

et al. 2020

Sci Rep

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A mutant form of the ataxin-1 protein with an expanded polyglutamine (polyQ) tract is the underlying cause of the inherited neurodegenerative disease spinocerebellar ataxia 1 (SCA1). In probing the biophysical features of the nuclear bodies (NBs) formed by polyQ-ataxin-1, we defined ataxin-1 NBs as spherical liquid protein/RNA droplets capable of rapid fusion. We observed dynamic exchange of the ataxin-1 protein into these NBs; notably, cell exposure to a pro-oxidant stress could trigger a transition to slower ataxin-1 exchange, typical of a hydrogel state, which no longer showed the same dependence on RNA or sensitivity to 1,6-hexanediol. Furthermore, we could alter ataxin-1 exchange dynamics either through modulating intracellular ATP levels, RNA helicase inhibition, or siRNA-mediated depletion of select RNA helicases. Collectively, these findings reveal the tunable dynamics of the liquid RNA/protein droplets formed by polyQ-ataxin-1.Within eukaryotic cells, complex biochemical reactions are facilitated by the concentration and restriction of key enzymes, substrates and regulators within well-defined membrane-bound organelles. In addition, numerous membrane-less intracellular compartments are important for a range of essential activities in both the cytoplasm (e.g. stress granules storing RNA molecules from stalled translation in response to environmental stresses 1 ) and the nucleus (e.g. nucleoli functioning in the biogenesis of ribosome subunits 2 , Cajal bodies in the processing and modification of short non-coding RNAs 3 , or promyelocytic leukemia (PML) bodies acting as sumoylation factories in response to interferon and oxidative stress 4 ). The formation of many of these membrane-less organelles is now understood to proceed via a phase separation process of specific constituent proteins, RNA and/or DNA molecules 5 . Thus, after a certain critical concentration threshold is exceeded, molecular assemblies of these constituents are formed with liquid-like behaviors that include fusing ability, viscous fluid dynamics, and high exchange rates with their surroundings in the nucleoplasm or cytoplasm 6-10 . This process of protein phase separation is now viewed as an essential mechanism for efficient compartmentalization that can be rapidly responsive to environmental challenges or intracellular changes 11,12 .Proteins that can undergo phase separation usually contain sequences conforming to either a low complexity region (LCR) or prion-like domain (PrLD) 8,11,13 ; these are protein domains typically with low amino acid diversity and little conformational heterogeneity 5,11 . These disordered structural characteristics can also contribute to an additional change known as protein phase transition, in which liquid-like condensates continue to become less dynamic and so form a more viscoelastic hydrogel or solid-like fibrous aggregates 12,14 . Many factors can promote the phase transition process such as time, pH, and altered amino acid sequences arising from gene mutations 8,9,15,16 . Thus, sequences typically co...

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Section: Down Regulation Of Dead/h-box Rna Helicases Decreases Ataxinmentioning

confidence: 99%

Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics

Zhang

Hinde

Schneider

et al. 2020

Sci Rep

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“…The MD intra-and interprotomer interactions are particularly interesting because they appear to exercise regulatory functions (1,19,28). For example, various single-site mutations in these segments potentiate Hsp104 activity (28)(29)(30). Cryo-EM results record a great deal of MD variability.…”

Section: Significancementioning

confidence: 99%

Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

Lin

Mayne

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.Hsp104 | hydrogen exchange | mass spectrometry | HX MS | HDX MS W e report an initial hydrogen exchange study of Hsp104, a large homohexameric member of the AAA+ superfamily (6 × 908 amino acid residues), in its various functional states. In Saccharomyces cerevisiae Hsp104 controls the prionogenesis and dissolution of the Sup35 translation termination factor (1). Hsp104 is interesting more generally for its ability to rescue aggregated proteins and even stably structured amyloids by mobilizing the driving energy of favorable ATP binding and hydrolysis to forcefully unfold proteins by threading them into or through its narrow central channel (1-5).Earlier low-resolution structures of Hsp104 and its bacterial homolog ClpB obtained by negative staining or cryo-electron microscopy (cryo-EM) showed symmetric flat hexamers (6-10). Recent higher resolution cryo-EM studies reveal more detailed global and fine-scale structural features, their dependence on the adenosine nucleotide that is bound, and enable mechanistic inferences (11,12). Thus, we now know what Hsp104 looks like, but the fundamental mechanisms and principles that determine how it works have so far been out of reach. The same is true for a rapidly growing number of other large protein molecules.Many methods have been used to measure changes and their connecting structural paths in allosteric systems, but there has been no way to connect site-resolved changes with site-resolved energetics and thus establish the importance and role of...

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“…To address this issue, we have engineered enhanced versions of Hsp104 that more effectively disaggregate disease-linked proteins such as α-syn (linked to PD), and TDP-43 and FUS (linked to ALS/FTD) under conditions where wild-type (WT) Hsp104 is ineffective Ryan et al, 2019;Tariq et al, 2019;Tariq et al, 2018;Torrente et al, 2016). Select potentiated variants reduce dopaminergic neurodegeneration in a C. elegans model of PD and reverse FUS aggregation and toxicity in mammalian cells (Yasuda et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Tuning Hsp104 specificity to selectively detoxify α-synuclein

Mack

Kim

Jackrel³

et al. 2020

Preprint

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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 that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, αsynuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms, involving α-synuclein disaggregation or detoxification of α-synuclein conformers without disaggregation. Importantly, both types of α-synucleinspecific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

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Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain

Cited by 40 publications

References 143 publications

Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics

Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics

Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

Tuning Hsp104 specificity to selectively detoxify α-synuclein

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