Potent Antiscrapie Activities of Degenerate Phosphorothioate Oligonucleotides

Kocisko, David A.; Vaillant, Andrew; Lee, Kil Sun; Arnold, Kevin; Bertholet, Nadine; Race, Richard E.; Olsen, Emily A.; Juteau, Jean-Marc; Caughey, Byron

doi:10.1128/aac.50.3.1034-1044.2006

Cited by 93 publications

(133 citation statements)

References 43 publications

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“…pentosan polysulfate (21), copper (18), suramin (30), and phosphorothioate oligonucleotides (22), affect the intracellular localization of PrP C . Because various synthetic cTPs inhibit PrP Sc formation, we wondered if hemin, as a natural cTP and potential physiological ligand for PrP C , can also affect PrP C localization.…”

Section: Resultsmentioning

confidence: 99%

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

Lee

Raymond

Schoen

et al. 2007

Journal of Biological Chemistry

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Hemin (iron protoporphyrin IX) is a crucial component of many physiological processes acting either as a prosthetic group or as an intracellular messenger. Some unnatural, synthetic porphyrins have potent anti-scrapie activity and can interact with normal prion protein (PrP C ). These observations raised the possibility that hemin, as a natural porphyrin, is a physiological ligand for PrP C . Accordingly, we evaluated PrP C interactions with hemin. When hemin (3-10 M) was added to the medium of cultured cells, clusters of PrP C formed on the cell surface, and the detergent solubility of PrP C decreased. The addition of hemin also induced PrP C internalization and turnover. The ability of hemin to bind directly to PrP C was demonstrated by hemin-agarose affinity chromatography and UV-visible spectroscopy. Multiple hemin molecules bound primarily to the N-terminal third of PrP C , with reduced binding to PrP C lacking residues 34 -94. These hemin-PrP C interactions suggest that PrP C may participate in hemin homeostasis, sensing, and/or uptake and that hemin might affect PrP C functions.

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Section: Resultsmentioning

confidence: 99%

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

Lee

Raymond

Schoen

et al. 2007

Journal of Biological Chemistry

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“…High pressure liquid chromatography-purified synthetic single-stranded RNA fragment 43-59 of SAF93 aptamer (SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] ) (22) and opRNA, a random 18-mer sequence, derived from a translational operator of MS2 bacteriophage (37) were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Nucleotide sequences were: SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] , 5Ј-GGA UGC AAU CUC CAU CCC-3Ј; and opRNA, 5Ј-AAA CAU GGG UUC CCA UGU-3Ј. Synthetic RNA samples were maintained lyophilized at Ϫ20°C and used in RNase-free water.…”

Section: Methodsmentioning

confidence: 99%

Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells

Gomes¹,

Millen²,

Ferreira³

et al. 2008

Journal of Biological Chemistry

123

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Conversion of the cellular prion protein (PrP C ) into its altered conformation, PrPSc , is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher ␤-sheet content and characteristics similar to those of PrP Sc . RNA molecules can also interact with PrP and potentially modulate PrP C to PrP Sc conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP C 3 PrP Sc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.Prion diseases can be infectious, sporadic, or inherited (1). Regardless of their origin, they are related to modifications of a ubiquitous membrane-anchored protein, the prion protein (PrP).3 Through a poorly understood process, the cellular PrP isoform (PrP C ), an ␣-helix-rich protein, undergoes a profound conformational change, acquiring higher ␤-sheet content; the latter isoform is known as PrP Sc (Sc from scrapie) and is the only known component of the infectious prion particle (1-4).The protein-only hypothesis postulates that PrP Sc "multiplies" by catalyzing the conversion of PrP C into a likeness of itself, thus becoming responsible for its own propagation (5). This hypothesis is based strongly on the fact that PrP knock-out mice are resistant to prion infection, suggesting that endogenous PrP is necessary for prion propagation and infection (6). It was also suggested, however, that an additional unknown factor could influence the PrP C to PrP Sc conversion (7-10). This molecule would act by lowering the free energy barrier between PrP C and PrP Sc and triggering conversion (11,12). In this field, a great number of biological macromolecules have emerged as candidates for conversion catalysts. Cellular adhesion molecules, nucleic acids (NAs), basal membrane molecules, and sulfated glycans, among other biological macromolecules, have been reported to interact with PrP C and to induce its conversion in...

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“…The recently reported crystal structure of human PrP C (24) and also the NMR structures of human PrP C show no obvious clefts for ligand binding (21). In contrast, a number of molecules have been demonstrated to bind to the unstructured region of PrP including pentosan polysulfate (25), Congo red (26), and a number of anionic tetrapyrroles (27,28). Congo red and anionic tetrapyrroles can stack in solution, and are believed to behave like polyanions and bind with low specificity to the N-terminal domain (29)(30)(31).…”

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confidence: 99%

Pharmacological chaperone for the structured domain of human prion protein

Nicoll¹,

Trevitt

Tattum

et al. 2010

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

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In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP C ). Numerous ligands have been reported to bind to human PrP C (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 1∶1 complex via the structured C terminus of huPrP with a K d of 4.5 ± 2 μM, which is in the range of its IC 50 for curing prion-infected cells of 1.6 ± 0.4 μM and the concentration required to inhibit protein-misfolding cyclic amplification. Therefore, this molecule tests the hypothesis that stabilization of huPrP C , as a principle, could be used in the treatment of human prion disease. The identification of a binding site with a defined 3D structure opens up the possibility of designing small molecules that stabilize huPrP and prevent its conversion into the disease-associated form.

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Potent Antiscrapie Activities of Degenerate Phosphorothioate Oligonucleotides

Cited by 93 publications

References 43 publications

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells

Pharmacological chaperone for the structured domain of human prion protein

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