The Extent of Protease Resistance of Misfolded Prion Protein Is Highly Dependent on the Salt Concentration

Concha‐Marambio, Luis; Díaz-Espinoza, Rodrigo; Soto, Claudio

doi:10.1074/jbc.m113.513267

Cited by 9 publications

(4 citation statements)

References 34 publications

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“…After treatment with increasing concentration of Proteinase K for 10 mins at 37 °C, the donor HEK293 cell lysate formed a smaller number of internalized Ago2 bodies with reduced a co-localization with PBs which were also getting significantly lowered in numbers with increasing concentration of Proteinase K treatment (Supplementary Figure S3A, S3D, S3E and S3F). This data suggests that the phase separated Ago2 are sensitive to Proteinase K. Therefore PB targeting is possibly reversible and unlike a prion-like transition, Ago2 remains protease sensitive after the phase separation to PBs (21). Compared to internalized Ago2 pool, the Proteinase K mediated degradation of FH-Ago2 present in the lysate suggests that the soluble protein in the lysate is similarly sensitive to proteinase K treatment compared to PB-phase separated FH-Ago2 (Supplementary Figure S3B and S3C).…”

Section: Resultsmentioning

confidence: 93%

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro

Ray,

Roychowdhury,

Chakraborty

et al. 2024

Preprint

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Phase separation to insoluble membrane-less organelles is a major way of activity regulation of specific proteins in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins are known to be localized to RNA-processing bodies, the subcellular structures which are formed due to assembly of several RNA binding and regulatory proteins in eukaryotic cells. Ago2 is the most important miRNA binding protein that by forming complex with miRNA binds to mRNAs having cognate miRNA binding sites and represses protein synthesis in mammalian cells. Factors which control compartmentalization of Ago2 and miRNA-repressed mRNAs to RNA processing bodies are largely unknown. We have adopted a detergent permeabilized cell-based assay system to follow the phase separation of exogenously added Ago2 to RNA processing bodiesin vitro. The Ago2 phase separation process is ATP dependent and is influenced by osmolarity and salt concentration of the reaction buffer. miRNA binding of Ago2 is essential for its targeting to RNA processing bodies and the compartmentalization process gets retarded by miRNA binding “sponge” protein HuR. This assay system found to be useful in identification of amyloid beta oligomers as miRNA-activity modulators which repress miRNA activity by enhancing Ago2-miRNP targeting to RNA processing bodies.Graphical AbstractmiRNA bound Ago2 gets phase separatedin vitroto RNA processing bodies (PBs) in detergent permeabilized mammalian cells.Phase separation of Ago2 to PBs is controlled by presence of ATP and RNA.Amyloid beta oligomers retard dynamics of Ago2 bodies to inhibit miRNA function and enhance PB targeting of Ago2 miRNPs.microRNA binding protein HuR can rescue Ago2 miRNP from PBs and inverse the effect of amyloid beta oligomers.

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Section: Resultsmentioning

confidence: 93%

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro

Ray,

Roychowdhury,

Chakraborty

et al. 2024

Preprint

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“…This is consistent with the suggestion that sPrP Sc may represent a breakdown product of rPrP Sc . 52 Interestingly, altering salt availability can also make rPrP Sc aggregates protease-sensitive, 53 again suggesting that sPrP Sc may represent a structural variant of rPrP Sc that is favored under certain biochemical conditions. Additionally, while small changes in pH are known to alter the conformation of PrP C and may also alter the conformation of PrP Sc , changes in pH can activate other proteins, such as chaperones 22 and proteases, 40 that may either directly alter the structure of PrP Sc or indirectly alter its structure by removing cofactors bound to PrP Sc .…”

Section: ■ Discussionmentioning

confidence: 99%

The Size and Stability of Infectious Prion Aggregates Fluctuate Dynamically during Cellular Uptake and Disaggregation

Shoup

Priola

2021

Biochemistry

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Prion diseases arise when PrP Sc , an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrP C , catalyzes the conversion of PrP C into more PrP Sc , which then accumulates in the brain leading to disease. PrP Sc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrP Sc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrP Sc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrP Sc aggregates from two different mouse-adapted prion strains, 22L, that can persistently infect cells and 87V, that cannot, changed during cellular uptake. We show that within the first 4 h following uptake large PrP Sc aggregates from both prion strains become less resistant to digestion by proteinase K (PK) through a mechanism that is dependent upon the acidic environment of endocytic vesicles. We further show that during disassembly, PrP Sc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrP Sc , with PrP Sc from the 87V strain disassembled more readily than PrP Sc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrP Sc from different prion strains change during cellular uptake and degradation, thereby potentially impacting the ability of prions to infect cells.

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“…Thus, it is considered the 'core' of the infectious agent and has been widely utilized for investigating self-assembly, aggregation and infectivity in prions [37][38][39]. It has been shown to misfold and aggregate under mild denaturing conditions into amyloid fibrils exhibiting 'scrapie' (PrP sc ) like characteristics [40,41]. The NMR structure of recombinant human PrP 90-231 (HuPrP) encompasses a disordered N-terminal region along with a structured, globular C-terminal domain (122-231) consisting of three α helices (α1, α2 and α3), two short antiparallel β-strands (β1 and β2) and two connecting loops (α2-α3 loop and β2-α2 loop) ( Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Modulation of prion polymerization and toxicity by rationally designed peptidomimetics

Srivastava

Sharma

Sadanandan

et al. 2016

Biochemical Journal

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Misfolding and aggregation of cellular prion protein is associated with a large array of neurological disorders commonly called the transmissible spongiform encephalopathies. Designing inhibitors against prions has remained a daunting task owing to limited information about mechanism(s) of their pathogenic self-assembly. Here, we explore the anti-prion properties of a combinatorial library of bispidine-based peptidomimetics (BPMs) that conjugate amino acids with hydrophobic and aromatic side chains. Keeping the bispidine unit unaltered, a series of structurally diverse BPMs were synthesized and tested for their prion-modulating properties. Administration of Leu- and Trp-BPMs delayed and completely inhibited the amyloidogenic conversion of human prion protein (HuPrP), respectively. We found that each BPM induced the HuPrP to form unique oligomeric nanostructures differing in their biophysical properties, cellular toxicities and response to conformation-specific antibodies. While Leu-BPMs were found to stabilize the oligomers, Trp-BPMs effected transient oligomerization, resulting in the formation of non-toxic, non-fibrillar aggregates. Yet another aromatic residue, Phe, however, accelerated the aggregation process in HuPrP. Molecular insights obtained through MD (molecular dynamics) simulations suggested that each BPM differently engages a conserved Tyr 169 residue at the α2-β2 loop of HuPrP and affects the stability of α2 and α3 helices. Our results demonstrate that this new class of molecules having chemical scaffolds conjugating hydrophobic/aromatic residues could effectively modulate prion aggregation and toxicity.

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The Extent of Protease Resistance of Misfolded Prion Protein Is Highly Dependent on the Salt Concentration

Cited by 9 publications

References 34 publications

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro

The Size and Stability of Infectious Prion Aggregates Fluctuate Dynamically during Cellular Uptake and Disaggregation

Modulation of prion polymerization and toxicity by rationally designed peptidomimetics

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