Contribution of two conserved glycine residues to fibrillogenesis of the 106-126 prion protein fragment. Evidence that a soluble variant of the 106-126 peptide is neurotoxic

The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, beta-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.     

Immunomodulation of the human prion peptide 106-126 aggregation

Site-directed monoclonal antibodies (mAbs) may interact with their antigens, leading to stabilization, refolding, and suppression of aggregation. In the following study, we show that mAbs raised against the peptide 106-126 of human prion protein (PrP 106-126) modulate the conformational changes occurring in the peptide exposed to aggregation conditions. MAbs 3-11 and 2-40 prevent PrP 106-126's fibrillar aggregation, disaggregates already formed aggregates, and inhibits the peptide's neurotoxic effect on the PC12 cells system, while mAb 3F4 has no protective effect. We suggest that there are key positions within the PrP 106-126 molecule where unfolding is initiated and their locking with specific antibodies may maintain the prion peptide native structure, reverse the aggregated peptide conformation, and lead to rearrangements involved in the essential feature of prion diseases.     

The Hydrophobic Core Sequence Modulates the Neurotoxic and Secondary Structure Properties of the Prion Peptide 106-126

The neurodegeneration seen in spongiform encephalopathies is believed to be mediated by protease-resistant forms of the prion protein (PrP). A peptide encompassing residues 106-126 of human PrP has been shown to be neurotoxic in vitro. The neurotoxicity of PrP106-126 appears to be dependent upon its adoption of an aggregated fibril structure. To examine the role of the hydrophobic core, AGAAAAGA, on PrP106-126 toxicity, we performed structure-activity analyses by substituting two or more hydrophobic residues for the hydrophilic serine residue to decrease its hydrophobicity. A peptide with a deleted alanine was also synthesized. We found all the peptides except the deletion mutant were no longer toxic on mouse cerebellar neuronal cultures. Circular dichroism analysis showed that the nontoxic PrP peptides had a marked decrease in beta-sheet structure. In addition, the mutants had alterations in aggregability as measured by turbidity, Congo red binding, and fibril staining using electron microscopy. These data show that the hydrophobic core sequence is important for PrP106-126 toxicity probably by influencing its assembly into a neurotoxic structure. The hydrophobic sequence may similarly affect aggregation and toxicity observed in prion diseases.     

In vivo cytotoxicity of the prion protein fragment 106-126

Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106-126 (P106-126) has been shown to be toxic in cultured hippocampal neurons (). Here, we show that P106-126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106-126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106-126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.     

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.     

Molecular dynamics simulations of alanine rich beta-sheet oligomers: Insight into amyloid formation

The aggregation observed in protein conformational diseases is the outcome of significant new beta-sheet structure not present in the native state. Peptide model systems have been useful in studies of fibril aggregate formation. Experimentally, it was found that a short peptide AGAAAAGA is one of the most highly amyloidogenic peptides. This peptide corresponds to the Syrian hamster prion protein (ShPrP) residues 113-120. The peptide was observed to be conserved in all species for which the PrP sequence has been determined. We have simulated the stabilities of oligomeric AGAAAAGA and AAAAAAAA (A8) by molecular dynamic simulations. Oligomers of both AGAAAAGA and AAAAAAAA were found to be stable when the size is 6 to 8 (hexamer to octamer). Subsequent simulation of an additional alpha-helical AAAAAAAA placed on the A8-octamer surface has revealed molecular events related to conformational change and oligomer growth. Our study addresses both the minimal oligomeric size of an aggregate seed and the mechanism of seed growth. Our simulations of the prion-derived 8-residue amyloidogenic peptide and its variant have indicated that an octamer is stable enough to be a seed and that the driving force for stabilization is the hydrophobic effect.     

Prion peptide 106-126 modulates the aggregation of cellular prion protein and induces the synthesis of potentially neurotoxic transmembrane PrP.

In infectious and familial prion disorders, neurodegeneration is often seen without obvious deposits of the scrapie prion protein (PrP(Sc)), the principal cause of neuronal death in prion disorders. In such cases, neurotoxicity must be mediated by alternative pathways of cell death. One such pathway is through a transmembrane form of PrP. We have investigated the relationship between intracellular accumulation of prion protein aggregates and the consequent up-regulation of transmembrane prion protein in a cell model. Here, we report that exposure of neuroblastoma cells to the prion peptide 106-126 catalyzes the aggregation of cellular prion protein to a weakly proteinase K-resistant form and induces the synthesis of transmembrane prion protein, the proposed mediator of neurotoxicity in certain prion disorders. The N terminus of newly synthesized transmembrane prion protein is cleaved spontaneously on the cytosolic face of the endoplasmic reticulum, and the truncated C-terminal fragment accumulates on the cell surface. Our results suggest that neurotoxicity in prion disorders is mediated by a complex pathway involving transmembrane prion protein and not by deposits of aggregated and proteinase K-resistant PrP alone.     

Effect of phospholipid liposomes on prion fragment (106–128) amyloid formation

Misfolding and aggregation of cellular prion protein (PrP^c) is a major molecular process involved in the pathogenesis of prion diseases. Here, we studied the aggregation properties of a prion fragment peptide PrP(106–128). The results show that the peptide aggregates in a concentration-dependent manner in an aqueous solution and that the aggregation is sensitive to pH and the preformed amyloid seeds. Furthermore, we show that the zwitterionic POPC liposomes moderately inhibit the aggregation of PrP(106–128), whereas POPC/cholesterol (8:2) vesicles facilitate peptide aggregation likely due to the increase of the lipid packing order and membrane rigidity in the presence of cholesterol. In addition, anionic lipid vesicles of POPG and POPG/cholesterol above a certain concentration accelerate the aggregation of the peptide remarkably. The strong electrostatic interactions between the N-terminal region of the peptide and POPG may constrain the conformational plasticity of the peptide, preventing insertion of the peptide into the inner side of the membrane and thus promoting fibrillation on the membrane surface. The results suggest that the charge properties of the membrane, the composition of the liposomes, and the rigidity of lipid packing are critical in determining peptide adsorption on the membrane surface and the efficiency of the membrane in catalyzing peptide oligomeric nucleation and amyloid formation. The peptide could be used as an improved model molecule to investigate the mechanistic role of the crucial regions of PrP in aggregation in a membrane-rich environment and to screen effective inhibitors to block key interactions between these regions and membranes for preventing PrP aggregation.     

Structural Conversion Rate Changes of Recombinant Bovine Prion by Designed Synthetic Peptides

An understanding of structural changes and self-assembly of proteins, which are thought to involve specific peptide?Cpeptide interactions, will contribute to the development of therapeutic agents and diagnosis for the detection of conformational diseases. We hypothesize that certain peptides may contribute to the conformational change of prion proteins. The present paper describes the discovery of prion-related synthetic peptides which influence structural conversion of recombinant bovine prion protein. The peptides designed are prion-protein fragments containing core domains consisting of ??-helical (human prion protein fragment 180?C195) and known ??-sheet (human prion protein fragment 169?C175) structures. Additionally several reported known ??-sheet breaker peptides and a conjugate consisting of ??-sheet and ??-helix segments based on the secondary structures of human prion protein, designated HPPSH, have been chemically synthesized by the conventional Fmoc solid-phase method and characterized by circular dichroism and the Thioflavin T fluorescence method. Our data indicated that the co-existence of peptides, HPPSH or other prion fragment peptides involving toxic core sequence (the fragment 106?C126), influenced the kinetic rate of aggregation and the lag-time of fibril formation of recombinant bovine prion protein except the core sequence itself. The method will be used for discovery of responsible material from natural resources. And designed peptides can be also used for bio-detection.     

Dissection of Conformational Conversion Events during Prion Amyloid Fibril Formation Using Hydrogen Exchange and Mass Spectrometry

A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH 7 has been characterized by hydrogen–deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen–deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~ 159–225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109–132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis.     

Aggregation of PrP106–126 on surfaces of neutral and negatively charged membranes studied by molecular dynamics simulations

Prion diseases are neurodegenerative disorders characterized by the aggregation of an abnormal form of prion protein. The interaction of prion protein and cellular membrane is crucial to elucidate the occurrence and development of prion diseases. Its fragment, residues 106–126, has been proven to maintain the pathological properties of misfolded prion and was used as a model peptide. In this study, explicit solvent molecular dynamics (MD) simulations were carried out to investigate the adsorption, folding and aggregation of PrP106–126 with different sizes (2-peptides, 4-peptides and 6-peptides) on the surface of both pure neutral POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and negatively charged POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) (3:1) lipids. MD simulation results show that PrP106–126 display strong affinity with POPC/POPG but does not interact with pure POPC. The positively charged and polar residues participating hydrogen bonding with membrane promote the adsorption of PrP106–126. The presence of POPC and POPC/POPG exert limited influence on the secondary structures of PrP106–126 and random coil structures are predominant in all simulation systems. Upon the adsorption on the POPC/POPG surface, the aggregation states of PrP106–126 have been changed and more small oligomers were observed. This work provides insights into the interactions of PrP106–126 and membranes with different compositions in atomic level, which expand our understanding the role membrane plays in the development of prion diseases. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.     

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.     

Prion Protein Peptide Neurotoxicity Can Be Mediated by Astrocytes

A peptide based on amino acids 106-126 of the sequence of human prion protein (PrP106-126) is neurotoxic in culture. A role for astrocytes mediating PrP106-126 toxicity was investigated. The toxicity of PrP106-126 to cerebellar cell cultures was reduced by aminoadipate, a gliotoxin. Normally, PrP106-126 is not toxic to cultures containing neurones deficient in the cellular isoform of prion protein (PrPc). However, PrP106-126 was toxic to cerebellar cells derived from Prnp(0/0) mice (deficient in PrPc expression) when those cerebellar cells were cocultured with astrocytes. This toxicity was found to occur only in the presence of PrPc-positive astrocytes and to be mediated by glutamate. Furthermore, PrPc-positive astrocytes were shown to protect Prnp(0/0) cerebellar cells from glutamate toxicity. This effect could be inhibited by PrP106-126. PrP106-126 did not enhance the toxicity of glutamate to neurones directly. When cerebellar cells were cocultured with astrocytes, the neurones became dependent on astrocytes for protection from glutamate toxicity and expressed an increased sensitivity to glutamate. In such a system, the protective effects of astrocytes against glutamate toxicity to neurones were inhibited by PrP106-126, resulting in a greater reduction in neuronal survival than would have been caused by PrP106-126 when astrocytes were not present. This new model provides a possible mechanism by which the gliosis in prion disease may accelerate the neurodegeneration seen in the later stages of the disease.     

DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.     

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP^Sc, an abnormally misfolded, protease-resistant, and β-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP^Sc and presents similar neurotoxicity as PrP^Sc. By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.     

Acetylcholinesterase triggers the aggregation of PrP 106-126

Acetylcholinesterase (AChE), a senile plaque component, promotes amyloid-beta-protein (Abeta) fibril formation in vitro. The presence of prion protein (PrP) in Alzheimer's disease (AD) senile plaques prompted us to assess if AChE could trigger the PrP peptides aggregation as well. Consequently, the efficacy of AChE on the PrP peptide spanning-residues 106-126 aggregation containing a coumarin fluorescence probe (coumarin-PrP 106-126) was studied. Kinetics of coumarin-PrP 106-126 aggregation showed a significant increase of maximum size of aggregates (MSA), which was dependent on AChE concentration. AChE-PrP 106-126 aggregates showed the tinctorial and optical amyloid properties as determined by polarized light and electronic microscopy analysis. A remarkable inhibition of MSA was obtained with propidium iodide, suggesting that AChE triggers PrP 106-126 and Abeta aggregation through a similar mechanism. Huprines (AChE inhibitors) also significantly decreased MSA induced by AChE as well, unveiling the potential interest for some AChE inhibitors as a novel class of potential anti-prion drugs.     

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH₂ species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.