Liquid-liquid phase separation and aggregation of the prion protein globular domain modulated by a high-affinity DNA aptamer

Matos, Carolina O.; Passos, Yulli M.; Amaral, Mariana J. do; Macedo, Bruno; Tempone, Matheus H.; Bezerra, Ohanna C. L.; Moraes, Milton Ozório; Almeida, Marcius S.; Weber, Gerald; Missailidis, Sotiris; Silva, Jerson L.; Pinheiro, Anderson S.; Cordeiro, Yraima

doi:10.1101/659037

Cited by 3 publications

(2 citation statements)

References 70 publications

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“…However, we also found evidence, by NMR, DLS, and observation by the naked eye, that the in vitro interaction between otherwise monomeric ASOs and monomeric PrP involves the formation of large aggregates, apparently containing both the ASO and PrP. Although we have not deeply characterized these aggregates to determine whether they are protease-resistant and/or represent liquid-liquid phase separation [28,29], our general findings are in line with several reports describing PrP aggregation in the presence of nucleic acids [28,[30][31][32][33]. Compounds that aggregate are often considered "pan-assay interference compounds" (PAINS) [23,34,35].…”

Section: Discussionmentioning

confidence: 77%

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides

et al. 2019

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Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP–ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO–PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

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Section: Discussionmentioning

confidence: 77%

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides

et al. 2019

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“…LLPS is increasingly recognized as a possible source of protein self-assembly into larger aggregates. In neurodegeneration, this has been most studied concerning RNA-binding proteins TDP43 and FUS, although LLPS also occurs in tau [81] and prion protein [82]. Recent studies show that for TDP43 to undergo LLPS as few as three tryptophan residues are required [83], however, minimum seeding unit required for efficient induction of TDP43 pathology is not yet ascertained.…”

Section: Tdp43mentioning

confidence: 99%

Invited Review: The role of prion‐like mechanisms in neurodegenerative diseases

Jaunmuktane

Brandner

2020

Neuropathology Appl Neurobio

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The prototype of transmissible neurodegenerative proteinopathies is prion diseases, characterized by aggregation of abnormally folded conformers of the native prion protein. A wealth of mechanisms has been proposed to explain the conformational conversion from physiological protein into misfolded, pathological form, mode of toxicity, propagation from cell-to-cell and regional spread. There is increasing evidence that other neurodegenerative diseases, most notably Alzheimer's disease (Ab and tau), Parkinson's disease (a-synuclein), frontotemporal dementia (TDP43, tau or FUS) and motor neurone disease (TDP43), exhibit at least some of the misfolded prion protein properties. In this review, we will discuss to what extent each of the properties of misfolded prion protein is known to occur for Ab, tau, a-synuclein and TDP43, with particular focus on self-propagation through seeding, conformational strains, selective cellular and regional vulnerability, stability and resistance to inactivation, oligomers, toxicity and summarize the most recent literature on transmissibility of neurodegenerative disorders.

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Liquid-liquid phase separation of full-length prion protein initiates conformational conversionin vitro

Tange

Ishibashi

Nakagaki

et al. 2020

Preprint

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What initiates the sporadic misfolding process of PrP C remains to be elucidated. Here, we show that rPrP undergoes LPS at the interface of ATPS of polyethylene glycol (PEG)/dextran; also, subsequent liquid-solid phase transition attributed to the maturation of β-sheet structure initiated the spontaneous conversion of rPrP into PrP Sc -like amyloid. The intrinsically disordered region of PrP, residues 23-89, and kosmotropic anions were essential for the overall reaction. As the conversion of the protein proceeds without providing kinetic energy in this in vitro model, the findings suggest that LPS is associated with the occurrence of prion diseases and provide a new understanding of prion emergence. AbstractIn prion diseases, the causative agent is thought to be solely composed of misfolded prion proteins (PrP Sc ). It has been shown that PrP Sc can convert host-encoded normal isoform of prion protein (PrP C ), but the spontaneous emergence of PrP Sc is still enigmatic. Certain amyloidogenic proteins have recently been shown to undergo liquid phase separation (LPS) and liquid-solid phase transition. These phenomena are now believed to play an important role in the initial step of pathological aggregation in neurodegenerative diseases. We investigated whether purified fulllength human recombinant prion protein (rPrP, residues: 23-231) refolded normally can directly convert into PrP Sc -like amyloid via LPS and subsequent phase transition. The proteins underwent LPS in an aqueous two-phase system (ATPS) composed of 9% polyethylene glycol (PEG; M.W. 6000) and 9% dextran (M.W. 180,000) with kosmotropic anions. The maturation of droplets could be monitored by microscopy because the droplets of rPrP were positively stained by thioflavin T (ThT) at the beginning of LPS. Notably, phase transition occurred within an hour, accompanied by the β-sheet transition of rPrP. The aged gels of rPrP acquired resistance to proteinase-K (PK) digestion and formed amyloids, as confirmed by Congo red staining. These overall reactions required the N-terminal region of PrP, amino acid residues 23-89, which is known as an intrinsically disordered region. Our results suggest that the aberrant phase transition may be associated with the sporadic emergence of prions, and our assay should be useful for understanding the in vitro generation of prions.

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Liquid-liquid phase separation and aggregation of the prion protein globular domain modulated by a high-affinity DNA aptamer

Cited by 3 publications

References 70 publications

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides

Invited Review: The role of prion‐like mechanisms in neurodegenerative diseases

Liquid-liquid phase separation of full-length prion protein initiates conformational conversionin vitro

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