Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.     

Chemically Induced Accumulation of GAGs Delays PrPSc Clearance but Prolongs Prion Disease Incubation Time

Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals. The only identified component of the infectious prion is PrP^Sc, an aberrantly folded isoform of PrP^C. Glycosaminoglycans, which constitute the main receptor for prions on cells, play a complex role in the pathogenesis of prion diseases. For example, while agents inducing aberrant lysosomal accumulation of GAGs such as Tilorone and Quinacrine significantly reduced PrP^Sc content in scrapie-infected cells, administration of Quinacrine to prion-infected subjects did not improve their clinical status. In this study, we investigated the association of PrP^Sc with cells cultured with Tilorone. We found that while the initial incorporation of PrP^Sc was similar in the treated and untreated cells, clearance of PrP^Sc from the Tilorone-treated cells was significantly impaired. Interestingly, prolonged administration of Tilorone to mice prior to prion infection resulted in a significant delay in disease onset, concomitantly with in vivo accumulation of lysosomal GAGs. We hypothesize that GAGs may complex with newly incorporated PrP^Sc in lysosomes and further stabilize the prion protein conformation. Over-stabilized PrP^Sc molecules have been shown to comprise reduced converting activity.     

Stereotypic behavior in Asiatic black and Malayan sun bears

The stereotypies of individually caged Asiatic black bears (Ursus thibetanus) and Malayan sun bears (Helarctos malayanus) were studied in detail. Stereotypies were performed by 27 of the 29 subjects, were primarily locomotory in form (e.g., pacing), and occupied on average 18% (standard error of the mean (SEM)=2.5) of daylight hours. Stereotypy levels during the night were almost negligible and were highly correlated with daytime levels. Total stereotypies peaked prior to food arrival, although oral stereotypies were most frequent after feeding. In general, stereotypies were performed in locations from which food arrival could be viewed, although Asiatic black bears were equally likely to exhibit stereotypy near a neighboring bear. Across individuals, stereotypy frequency was inversely correlated with inactivity and increased with age. Older bears also showed less normal activity and a reduced diversity of normal behavior. Stereotypy levels were unrelated to levels of “compulsive” behavior (e.g., hair plucking) or repetitive self‐sucking–a potential deprivation stereotypy. More frequent stereotypies were performed more invariantly (i.e., were more predictable from one repetition to the next) and in more diverse contexts, namely 1) outside the pre‐feeding period, and 2) during the night. Contrary to observations reported elsewhere, higher frequencies of stereotypy were not associated with reduced behavioral diversity, or with a more elaborate repertoire of stereotypy forms and sequences. Although the two species did not differ in overall frequency, the stereotypies of sun bears appeared to be more food‐motivated than those of Asiatic black bears: the sun bears displayed a higher frequency and diversity of oral stereotypies, and higher levels of pre‐feeding stereotypy, and performed significantly more of their total stereotypies in locations from which they could view food arrival. This study demonstrates how analyzing stereotypies in detail can help identify the motivations that underlie these behaviors, and potentially reveal their degree of establishment–both of which are important factors in stereotypy treatment. Zoo Biol 23:409–430, 2004. © 2004 Wiley‐Liss, Inc.     

Structural Modifications of the Female Gametophyte Induced by Temperature in Nicotiana tabacum

Effect of environmental conditions on formation of Nicotiana tabacum L. megagametophyte was studied. It was established, that unfavorable temperatures can specifically modify the structure of embryo sacs (ES). At low temperature (9/5 °C), ES with a reduced number of cells or with egg-like synergide(s) can be formed; at high variable (40/25 °C) or constant (37 °C) temperatures, ES with excessive numbers of cells or with synergide-like egg cells arise. Total frequencies of the changed ES patterns varied from 8 up to 35 % per plant and depended on the plant genotype and conditions of exposure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Successful feeding enrichment for captive chimpanzees

An analysis of a feeding enrichment program for captive chimpanzees was completed to determine the effects of that program on the incidence of agonistic and abnormal behaviors by using 41 adult chimpanzees in six social groups. Four feeding enrichment procedures were implemented simultaneously in the experimental phase of this ABA design study. The procedures were planned to elicit time-consuming, noncompetitive feeding, theorizing a consequent reduction in agonism and detrimental abnormal behaviors typically occurring in captive chimpanzees. The 270 hours of data were collected over a 6-month period. A multivariate analysis of variance result supported the hypothesis, as the barrage of feeding enrichment techniques significantly reduced agonism and abnormal behaviors. This study's significance lies in its application of the scientific method to improving the husbandry of captive primates.     

Naturally prion resistant mammals

Each known abnormal prion protein (PrP^Sc) is considered to have a specific range and therefore the ability to infect some species and not others. Consequently, some species have been assumed to be prion disease resistant as no successful natural or experimental challenge infections have been reported. This assumption suggested that, independent of the virulence of the PrP^Sc strain, normal prion protein (PrP^C) from these ‘resistant’ species could not be induced to misfold. Numerous in vitro and in vivo studies trying to corroborate the unique properties of PrP^Sc have been undertaken. The results presented in the article “Rabbits are not resistant to prion infection” demonstrated that normal rabbit PrP^C, which was considered to be resistant to prion disease, can be misfolded to PrP^Sc and subsequently used to infect and transmit a standard prion disease to leporids. Using the concept of species resistance to prion disease, we will discuss the mistake of attributing species specific prion disease resistance based purely on the absence of natural cases and incomplete in vivo challenges. The BSE epidemic was partially due to an underestimation of species barriers. To repeat this error would be unacceptable, especially if present knowledge and techniques can show a theoretical risk. Now that the myth of prion disease resistance has been refuted it is time to re-evaluate, using the new powerful tools available in modern prion laboratories, whether any other species could be at risk.     

ERp57 as a novel cellular factor controlling prion protein biosynthesis: Therapeutic potential of protein disulfide isomerases

Disturbance of endoplasmic reticulum (ER) proteostasis is observed in Prion-related disorders (PrDs). The protein disulfide isomerase ERp57 is a stress-responsive ER chaperone up-regulated in the brain of Creutzfeldt-Jakob disease patients. However, the actual role of ERp57 in prion protein (PrP) biogenesis and the ER stress response remained poorly defined. We have recently addressed this question using gain- and loss-of-function approaches in vitro and animal models, observing that ERp57 regulates steady-state levels of PrP. Our results revealed that ERp57 modulates the biosynthesis and maturation of PrP but, surprisingly, does not contribute to the global cellular reaction against ER stress in neurons. Here we discuss the relevance of ERp57 as a possible therapeutic target in PrDs and other protein misfolding disorders.     

Helix-coil transition of PrP106-126: molecular dynamic study.

A set of 34 molecular dynamic (MD) simulations totaling 305 ns of simulation time of the prion protein-derived peptide PrP106-126 was performed with both explicit and implicit solvent models. The objective of these simulations is to investigate the relative stability of the alpha-helical conformation of the peptide and the mechanism for conversion from the helix to a random-coil structure. At neutral pH, the wild-type peptide was found to lose its initial helical structure very fast, within a few nanoseconds (ns) from the beginning of the simulations. The helix breaks up in the middle and then unwinds to the termini. The spontaneous transition into the random coil structure is governed by the hydrophobic interaction between His(111) and Val(122). The A117V mutation, which is linked to GSS disease, was found to destabilize the helix conformation of the peptide significantly, leading to a complete loss of helicity approximately 1 ns faster than in the wild-type. Furthermore, the A117V mutant exhibits a different mechanism for helix-coil conversion, wherein the helix begins to break up at the C-terminus and then gradually to unwind towards the N-terminus. In most simulations, the mutation was found to speed up the conversion through an additional hydrophobic interaction between Met(112) and the mutated residue Val(117), an interaction that did not exist in the wild-type peptide. Finally, the beta-sheet conformation of the wild-type peptide was found to be less stable at acidic pH due to a destabilization of the His(111)-Val(122), since at acidic pH this histidine is protonated and is unlikely to participate in hydrophobic interaction.