A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity

Fare, Charlotte M.; Rhine, Kevin; Lam, Andrew K. C.; Myong, Sua; Shorter, James

doi:10.1016/j.jbc.2022.102806

Cited by 13 publications

(20 citation statements)

References 135 publications

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“…To this end, we utilized a human (HeLa) cell model by expressing mCherry-tagged Hsp104 and GFP- tagged FUS in HeLa cells. 80 We selected a panel of potentiated variants with minimal off-target toxicity to assess in this system. Importantly, potentiated Hsp104 variants can significantly suppress FUS cytoplasmic mislocalization and aggregation, whereas Hsp104 cannot (Figure 7A).…”

Section: Resultsmentioning

confidence: 99%

Design principles to tailor Hsp104 therapeutics

Lin,

Carman,

Gambogi

et al. 2024

Preprint

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The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics.

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Section: Resultsmentioning

confidence: 99%

Design principles to tailor Hsp104 therapeutics

Lin,

Carman,

Gambogi

et al. 2024

Preprint

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Section: Resultsmentioning

confidence: 99%

“…We omitted the C-terminal RGG domain and PY-NLS to prevent spontaneous phase separation of full-length FUS proteins with mutated RNA-binding regions, 34 and also to reduce interaction with the nuclear-import receptor, Karyopherin-b2, which can prevent FUS phase separation. 8,[40][41][42][43] We exposed cells co-expressing FUS-SspB constructs and photo-activatable seeds (iLID-EGFP-FTH1) 39 to acute (30 second) blue light activation sequences and assessed FUS condensate formation and dissolution (Figure 4B). Interestingly, mutations within the RRM region (4FL, red trace) led to enhanced formation of light-induced FUS-SspB condensates compared to wild-type (WT) FUS1-453 (black trace), whereas mutations within the ZnF domain (4CA, blue trace) only mildly increased light-induced phase separation (LIPS) (Figure 4B, C).…”

Section: The Fus Rrm and Znf Domains Cooperate To Maintain Fus Solubi...mentioning

confidence: 99%

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains

Guo,

Mann,

Mauna

et al. 2023

Preprint

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RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

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“…For this reason, single-component disaggregases may prove to be more tractable therapeutically. 70,72,101–108…”

Section: Discussionmentioning

confidence: 99%

Defining a small-molecule stimulator of the human Hsp70-disaggregase system with selectivity for DnaJB proteins

Chuang,

Cupo,

Bevan

et al. 2024

Preprint

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Hsp70, Hsp40, and Hsp110 form a human protein-disaggregase system that solubilizes and reactivates proteins trapped in aggregated states. However, this system fails to maintain proteostasis in fatal neurodegenerative diseases. Here, we potentiate the human Hsp70-disaggregase system pharmacologically. By scouring a collection of dihydropyrimidines, we disambiguate a small molecule that specifically stimulates the Hsp70-disaggregase system against disordered aggregates and α-synuclein fibrils. The newly identified lead compound stimulates the disaggregase activity of multiple active human Hsp70, Hsp40, Hsp110 chaperone sets, with selectivity for combinations that include DnaJB1 or DnaJB4 as the Hsp40. We find that the relative stoichiometry of Hsp70, Hsp40, and Hsp110 dictates disaggregase activity. Remarkably, our lead compound shifts the composition of active chaperone stoichiometries by preferentially activating combinations with lower DnaJB1 concentrations. Our findings unveil a small molecule that stimulates the Hsp70-disaggregase system, even at suboptimal chaperone stoichiometries, which could be developed for the treatment of neurodegenerative diseases.

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A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity

Cited by 13 publications

References 135 publications

Design principles to tailor Hsp104 therapeutics

Design principles to tailor Hsp104 therapeutics

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains

Defining a small-molecule stimulator of the human Hsp70-disaggregase system with selectivity for DnaJB proteins

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