Factors affecting interactions between prion protein isoforms

2002 ◽  
Vol 30 (4) ◽  
pp. 565-569 ◽  
Author(s):  
B. Caughey ◽  
G. S. Baron
Keyword(s):  
Prion Protein ◽  
Recipient Cell ◽  
Transmissible Spongiform Encephalopathy ◽  
Heparan Sulphate ◽  
Protein Isoforms ◽  
Spongiform Encephalopathy ◽  
Factors Affecting ◽  
C Terminus ◽  
Low Concentrations

Interactions between normal, protease-sensitive prion protein (PrP-sen or PrPc) and its protease-resistant isoform (PrP-res or PrPsc) are critical in transmissible spongiform encephalopathy (TSE) diseases. To investigate the propagation of PrP-res between cells we tested whether PrP-res in scrapie brain microsomes can induce the conversion of PrP-sen to PrP-res if the PrP-sen is bound to uninfected raft membranes. Surprisingly, no conversion was observed unless the microsomal and raft membranes were fused or PrP-sen was released from raft membranes. These results suggest that the propagation of infection between cells requires transfer of PrP-res into the membranes of the recipient cell. To assess potential cofactors in PrP conversion, we used cell-free PrP conversion assays to show that heparan sulphate can stimulate PrP-res formation, supporting the idea that endogenous sulphated glycosaminoglycans can act as important cofactors or modulators of PrP-res formation in vivo. In an effort to develop therapeutics, the antimalarial drug quinacrine was identified as an inhibitor of PrP-res formation in scrapie-infected cell cultures. Confirmation of the latter result by others has led to the initiation of human clinical trials as a treatment for Creutzfeldt-Jakob disease. PrP-res formation can also be inhibited using a variety of other types of small molecule, specific synthetic PrP peptides, and an antiserum directed at the C-terminus of PrP-sen. The latter results help to localize the sites of interaction between PrP-sen and PrP-res. Disruption of those interactions with antibodies or peptidomimetic drugs may be an attractive therapeutic strategy. The likelihood that PrP-res inhibitors can rid TSE-infected tissues of PrP-res would presumably be enhanced if PrP-res formation were reversible. However, our attempts to measure dissociation of PrP-sen from PrP-res have failed under non-denaturing conditions. Finally, we have attempted to induce the spontaneous conversion of PrP-sen into PrP-res using low concentrations of detergents. A conformational conversion from α-helical monomers into high-β-sheet aggregates and fibrils was induced by low concentrations of the detergent sarkosyl; however, the aggregates had neither infectivity nor the characteristic protease-resistance of PrP-res.

scholarly journals PrP P102L and Nearby Lysine Mutations Promote Spontaneous In Vitro Formation of Transmissible Prions

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Allison Kraus ◽  
Gregory J. Raymond ◽  
Brent Race ◽  
Katrina J. Campbell ◽  
Andrew G. Hughson ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Transmissible Spongiform Encephalopathy ◽  
Clinical Signs ◽  
Protein Aggregates ◽  
Structural Features ◽  
Spongiform Encephalopathy ◽  
Specific Sequence

ABSTRACT Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo. To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro. Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids. IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.

scholarly journals Characterization and polyanion-binding properties of purified recombinant prion protein

1999 ◽  
Vol 342 (3) ◽  
pp. 605-613 ◽  
Author(s):  
Debbie B. BRIMACOMBE ◽  
Alan D. BENNETT ◽  
Fred S. WUSTEMAN ◽  
Andrew C. GILL ◽  
Janine C. DANN ◽  
...  
Keyword(s):  
Prion Protein ◽  
Mammalian Cells ◽  
Metal Ion ◽  
Transmissible Spongiform Encephalopathy ◽  
Spongiform Encephalopathy ◽  
Divalent Metal Ions ◽  
Dissociation Kinetics ◽  
Recombinant Prion Protein

Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein.

scholarly journals Inhibition of Protease-Resistant Prion Protein Accumulation In Vitro by Curcumin

2003 ◽  
Vol 77 (9) ◽  
pp. 5499-5502 ◽  
Author(s):  
Byron Caughey ◽  
Lynne D. Raymond ◽  
Gregory J. Raymond ◽  
Laura Maxson ◽  
Jay Silveira ◽  
...  
Keyword(s):  
Prion Protein ◽  
Inhibitory Concentration ◽  
Transmissible Spongiform Encephalopathy ◽  
Neuroblastoma Cells ◽  
In Vivo Studies ◽  
Spongiform Encephalopathy ◽  
Protein Accumulation ◽  
The Brain

ABSTRACT Inhibition of the accumulation of protease-resistant prion protein (PrP-res) is a prime strategy in the development of potential transmissible spongiform encephalopathy (TSE) therapeutics. Here we show that curcumin (diferoylmethane), a major component of the spice turmeric, potently inhibits PrP-res accumulation in scrapie agent-infected neuroblastoma cells (50% inhibitory concentration, ∼10 nM) and partially inhibits the cell-free conversion of PrP to PrP-res. In vivo studies showed that dietary administration of curcumin had no significant effect on the onset of scrapie in hamsters. Nonetheless, other studies have shown that curcumin is nontoxic and can penetrate the brain, properties that give curcumin advantages over inhibitors previously identified as potential prophylactic and/or therapeutic anti-TSE compounds.

scholarly journals Species-Independent Inhibition of Abnormal Prion Protein (PrP) Formation by a Peptide Containing a Conserved PrP Sequence

1999 ◽  
Vol 73 (8) ◽  
pp. 6245-6250 ◽  
Author(s):  
Joëlle Chabry ◽  
Suzette A. Priola ◽  
Kathy Wehrly ◽  
Jane Nishio ◽  
James Hope ◽  
...  
Keyword(s):  
Prion Protein ◽  
Transmissible Spongiform Encephalopathy ◽  
Neuroblastoma Cell ◽  
Spongiform Encephalopathy ◽  
Inhibitory Effects ◽  
Mouse Neuroblastoma Cell ◽  
Mouse Neuroblastoma ◽  
Scrapie Strain ◽  
Species Specific ◽  
Scrapie Strains

ABSTRACT Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.

scholarly journals Evaluation of Quinacrine Treatment for Prion Diseases

2003 ◽  
Vol 77 (15) ◽  
pp. 8462-8469 ◽  
Author(s):  
A. Barret ◽  
F. Tagliavini ◽  
G. Forloni ◽  
C. Bate ◽  
M. Salmona ◽  
...  
Keyword(s):  
Prion Protein ◽  
De Novo ◽  
Neuroblastoma Cells ◽  
Animal Origin ◽  
Detectable Effect ◽  
Spongiform Encephalopathy ◽  
Protease Resistance ◽  
New Variant

ABSTRACT Based on in vitro observations in scrapie-infected neuroblastoma cells, quinacrine has recently been proposed as a treatment for Creutzfeldt-Jakob disease (CJD), including a new variant CJD which is linked to contamination of food by the bovine spongiform encephalopathy (BSE) agent. The present study investigated possible mechanisms of action of quinacrine on prions. The ability of quinacrine to interact with and to reduce the protease resistance of PrP peptide aggregates and PrPres of human and animal origin were analyzed, together with its ability to inhibit the in vitro conversion of the normal prion protein (PrPc) to the abnormal form (PrPres). Furthermore, the efficiencies of quinacrine and chlorpromazine, another tricyclic compound, were examined in different in vitro models and in an experimental murine model of BSE. Quinacrine efficiently hampered de novo generation of fibrillogenic prion protein and PrPres accumulation in ScN2a cells. However, it was unable to affect the protease resistance of preexisting PrP fibrils and PrPres from brain homogenates, and a “curing” effect was obtained in ScGT1 cells only after lengthy treatment. In vivo, no detectable effect was observed in the animal model used, consistent with other recent studies and preliminary observations in humans. Despite its ability to cross the blood-brain barrier, the use of quinacrine for the treatment of CJD is questionable, at least as a monotherapy. The multistep experimental approach employed here could be used to test new therapeutic regimes before their use in human trials.

scholarly journals Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein

2006 ◽  
Vol 87 (12) ◽  
pp. 3753-3761 ◽  
Author(s):  
Martin Eiden ◽  
Gottfried J. Palm ◽  
Winfried Hinrichs ◽  
Ulrich Matthey ◽  
Ralph Zahn ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
De Novo ◽  
Proteinase K ◽  
Spongiform Encephalopathy ◽  
Cooperative Action ◽  
Specific Effects ◽  
Scrapie Strains ◽  
Recombinant Prion Protein

This study describes the conversion of murine PrPC by PrPSc from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrPres. Using this assay, newly formed PrPres could be detected by an antibody that discriminated de novo PrPres and the original PrPSc seed. The results suggested that PrPres formation occurs in three phases: the first 48 h when PrPres formation is delayed, followed by a period of substantially accelerated PrPres formation and a plateau phase when a maximum concentration of PrPres is reached after 72 h. The conversion of prokaryotically expressed PrPC by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrPres fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrPC was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrPres fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrPres. This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.

scholarly journals Prion Type-Dependent Deposition ofPRNPAllelic Products in Heterozygous Sheep

2015 ◽  
Vol 90 (2) ◽  
pp. 805-812 ◽  
Author(s):  
J. P. M. Langeveld ◽  
J. G. Jacobs ◽  
N. Hunter ◽  
L. J. M. van Keulen ◽  
F. Lantier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
Transmissible Spongiform Encephalopathy ◽  
Infected Sheep ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Sequence Variants ◽  
Spongiform Encephalopathies

ABSTRACTSusceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triplet represents the amino acids at codons 136, 154, and 171, respectively) sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In bovine spongiform encephalopathy (BSE) infection, the polymorphism effect is quite different although the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease-resistant prion protein (PrPres) from brain of heterozygous ARR/VRQ scrapie-infected sheep was compared with that of BSE-infected sheep with a similar genotype. A triplex Western blotting technique was used to estimate the two allotype PrP fractions in PrPresmaterial from BSE-infected ARR/VRQ sheep. PrPresin BSE contained equimolar amounts of VRQ- and ARR-PrP, which contrasts with the excess (>95%) VRQ-PrP fraction found in PrP in scrapie. This is evidence that transmissible spongiform encephalopathy (TSE) agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state), determine the polymorphic dependence of the PrPresaccumulation process in prion formation as well as the disease-associated phenotypic expressions in the host.IMPORTANCETransmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts, as has been shown for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie-infected sheep that produce two PrP variants associated with opposite scrapie susceptibilities (136V-PrP variant, high; 171R-PrP variant, very low) contain in their prion material over 95% of the 136V PrP variant. However, when these sheep are infected with prions from cattle (bovine spongiform encephalopathy [BSE]), both PrP variants occur in equal ratios. This shows that the infecting prion type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

scholarly journals Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease

2006 ◽  
Vol 80 (2) ◽  
pp. 596-604 ◽  
Author(s):  
Gregory J. Raymond ◽  
Emily A. Olsen ◽  
Kil Sun Lee ◽  
Lynne D. Raymond ◽  
P. Kruger Bryant ◽  
...  
Keyword(s):  
Cell Line ◽  
Prion Protein ◽  
Chronic Wasting Disease ◽  
Transmissible Spongiform Encephalopathy ◽  
Mule Deer ◽  
Simian Virus 40 ◽  
Spongiform Encephalopathy ◽  
Wasting Disease ◽  
Neuronal Marker

ABSTRACT Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrPCWD) was used as an indicator of CWD infection. Although no PrPCWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrPCWD-positive clone out of 51. This clone, designated MDBCWD, has maintained stable PrPCWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrPCWD-positive subclones out of 30, one of which was designated MDBCWD2. The MDBCWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrPCWD accumulation in MDBCWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrPCWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

scholarly journals Transmission and Adaptation of Chronic Wasting Disease to Hamsters and Transgenic Mice: Evidence for Strains

2007 ◽  
Vol 81 (8) ◽  
pp. 4305-4314 ◽  
Author(s):  
Gregory J. Raymond ◽  
Lynne D. Raymond ◽  
Kimberly D. Meade-White ◽  
Andrew G. Hughson ◽  
Cynthia Favara ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Prion Protein ◽  
Chronic Wasting Disease ◽  
Transmissible Spongiform Encephalopathy ◽  
Wild Type Mouse ◽  
Mouse Strains ◽  
Spongiform Encephalopathy ◽  
Wild Type ◽  
Wasting Disease ◽  
Incubation Periods

ABSTRACT In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.

Sign in / Sign up

Export Citation Format

Share Document