Substoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of Sup35 prion domain

Mahapatra, S.; Sarbahi, Anusha; Madhu, Priyanka; Swasthi, Hema M.; Sharma, Abhishek; Singh, Priyanka; Mukhopadhyay, Samrat

doi:10.1016/j.jbc.2022.102143

Cited by 3 publications

(7 citation statements)

References 64 publications

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“…First, the extensive binding of ATP in the lysine-rich M-domain of NM reinforced an extensive crosslinking through ATP molecules, especially in high concentrations, that resulted in a protease-resistant core. This remarkable conformational compactness probably made these amyloids inaccessible to disaggregating factors such as free ATP or Hsp104 that target the M-domain ( 25 , 51 ). Second, a remodeling in amyloid conformations in NM-ATP amyloids because of the binding of ATP during aggregation.…”

Section: Discussionmentioning

confidence: 99%

“…According to this mechanism, preformed amyloid entities known as seeds can be transmitted to the neighboring healthy cells, where they can template amyloid formation ( 21 ). Apart from the formation, the fragmentation of high molecular weight fibrils by cellular disaggregases is critical to generate growth-competent amyloids that facilitate prion-like propagation ( 22 , 23 , 24 , 25 ). Such fragmented fibrils and oligomers exhibit higher permeability through lipid bilayers of both donor and acceptor cells and are considered as the predominant species for infection and toxicity across amyloid-associated diseases ( 26 , 27 , 28 ).…”

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confidence: 99%

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ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Mahapatra¹,

Sarbahi²,

Punia³

et al. 2023

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Mahapatra¹,

Sarbahi²,

Punia³

et al. 2023

Journal of Biological Chemistry

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“…Firstly, the extensive binding of ATP in the lysine-rich M-domain of NM reinforced an extensive crosslinking through ATP molecules, especially in high concentrations, that resulted in a protease-resistant core. This remarkable conformational compactness probably made these amyloids inaccessible to disaggregating factors such as free ATP or Hsp104 that target the M-domain (25)(51). Secondly, a remodeling in amyloid conformations in NM-ATP amyloids due to the binding of ATP during aggregation.…”

Section: Discussionmentioning

confidence: 99%

“…C-terminal hexa-histidine tagged recombinant Sup35NM protein was overexpressed in BL21 (DE3)/pLysS cells using IPTG, and then from harvested cells, proteins were extracted; the extracted proteins were subjected to first Ni-NTA purification by applying a gradient of imidazole and further from a Q-sepharose column using the gradient of sodium chloride. The detailed protocol was previously described by (25)(55). Also, for the single cysteine mutant of NM, the purification remains the same as mentioned above with the addition of β-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

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ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Mahapatra

Sarbahi

Punia

et al. 2022

Preprint

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Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. Here, we demonstrate that ATP modulates the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase-separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP dose independently disaggregates preformed NM fibrils. Furthermore, high concentrations of ATP delimited the number of seeds by generating compact, ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase. Additionally, (low) pathological ATP concentrations restricted autocatalytic amplification by forming structurally distinct seeding-inefficient amyloids. Our results provide mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions.

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Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-β42 aggregation and fibril formation

Ghosh

Tugarinov

Clore

2023

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

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The chaperone Hsp104, a member of the Hsp100/Clp family of translocases, prevents fibril formation of a variety of amyloidogenic peptides in a paradoxically substoichiometric manner. To understand the mechanism whereby Hsp104 inhibits fibril formation, we probed the interaction of Hsp104 with the Alzheimer’s amyloid-β42 (Aβ42) peptide using a variety of biophysical techniques. Hsp104 is highly effective at suppressing the formation of Thioflavin T (ThT) reactive mature fibrils that are readily observed by atomic force (AFM) and electron (EM) microscopies. Quantitative kinetic analysis and global fitting was performed on serially recorded 1 H- 15 N correlation spectra to monitor the disappearance of Aβ42 monomers during the course of aggregation over a wide range of Hsp104 concentrations. Under the conditions employed (50 μM Aβ42 at 20 °C), Aβ42 aggregation occurs by a branching mechanism: an irreversible on-pathway leading to mature fibrils that entails primary and secondary nucleation and saturating elongation; and a reversible off-pathway to form nonfibrillar oligomers, unreactive to ThT and too large to be observed directly by NMR, but too small to be visualized by AFM or EM. Hsp104 binds reversibly with nanomolar affinity to sparsely populated Aβ42 nuclei present in nanomolar concentrations, generated by primary and secondary nucleation, thereby completely inhibiting on-pathway fibril formation at substoichiometric ratios of Hsp104 to Aβ42 monomers. Tight binding to sparsely populated nuclei likely constitutes a general mechanism for substoichiometric inhibition of fibrillization by a variety of chaperones. Hsp104 also impacts off-pathway oligomerization but to a much smaller degree initially reducing and then increasing the rate of off-pathway oligomerization.

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Substoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of Sup35 prion domain

Cited by 3 publications

References 64 publications

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification

Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-β42 aggregation and fibril formation

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