Molecular dynamics study of the fibril elongation of the prion protein fragment PrP106-126

The present paper aims at exploring the elongation of the PrP106-126 fibril under acid environments through molecular dynamics simulation. It shows that influenced by the edge strands of the fibril, single PrP106-126 peptide forms beta-sheet and becomes a new element of the fibril. Under acidic condition, single PrP106-126 fragment presents a much larger variety of conformations than it does under neural condition. However, acidic condition does not largely affect the stability of the PrP106-126 fibril. Consequently, the speed of the fibril elongation can be dramatically increased by lowering the pH value of the solution. The pH values are adjusted by either altering the protonation state of the residues or adding hydronium ions or hydroxyl ions.     

多肽PrP106-126对培养神经细胞朊蛋白基因表达的影响

神经细胞是传染性海绵状脑病(transmissible spongiform encephalopathies, TSEs)的重要靶细胞,PrP106-126是研究TSEs致病机理的理想工具,对PrP106-126作用的培养神经细胞模型进行研究,有利于了解朊蛋白的功能和探讨TSEs的分子致病机制.本研究利用PrP106-126构建了大脑皮质和小脑颗粒神经元作用模型,对神经细胞的存活和朊蛋白基因的表达进行了研究.结果表明PrP106-126作用于培养神经细胞导致其存活率的显著下降;大脑皮质神经元经PrP 106-126处理后,与SCR处理组和对照组相比,基因表达的量明显下降,处理后的小脑颗粒神经元也有类似的情况出现,两者之间下降的幅度和时间不同.我们的研究结果为研究朊蛋白在TSEs发生中的作用和深入了解TSE的分子致病机制提供了基础数据.     

多肽PrP106—126对培养神经细胞朊蛋白基因表达的影响

神经细胞是传染性海绵状脑病（transmissible spongiform encephalopathies,TSEs）的重要靶细胞,PrP106-126是研究TSEs致病机理的理想工具,对PrP106-126作用的培养神经细胞模型进行研究,有利于了解朊蛋白的功能和探讨TSEs的分子致病机制。本研究利用PrP106-126构建了大脑皮质和小脑颗粒神经元作用模型,对神经细胞的存活和朊蛋白基因的表达进行了研究。结果表明：PrP106-126作用于培养神经细胞导致其存活率的显著下降;大脑皮质神经元经PrP106-126处理后,与SCR处理组和对照组相比,基因表达的量明显下降,处理后的小脑颗粒神经元也有类似的情况出现,两者之间下降的幅度和时间不同。我们的研究结果为研究朊蛋白在TSEs发生中的作用和深入了解TSE的分子致病机制提供了基础数据。     

Antiaggregating antibody raised against human PrP 106-126 recognizes pathological and normal isoforms of the whole prion protein

Antibodies to the prion protein (PrP) have been critical to the neuropathological and biochemical characterization of PrP-related degenerative diseases in humans and animals. Although PrP is highly conserved evolutionarily, there is some sequence divergence among species; as a consequence, anti-PrP antibodies have a wide spectrum of reactivity when challenged with PrP from diverse species. We have produced an antibody [monoclonal antibody (mAb) 2-40] raised against a synthetic peptide corresponding to residues (106-126 of human PrP and have characterized it by epitope mapping, Western immunoblot analysis, and immunohistochemistry. The antibody recognizes not only human PrP isoforms but also pathological PrP from all species tested (i.e., sheep, hamsters, and mice). Together with the fact that it recognizes the whole PrP in both cellular and scrapie isoforms, mAb 2-40 may be helpful in studying conformational changes of the PrP, as well as establishing a possible connection between human and animal diseases.     

PrP 106-126 Altered PrP mRNA gene expression in mouse microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106–126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106–126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106–126, and indicate that microglial cells might play a critical role in prion pathogenesis.     

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.     

Interaction between 14-3-3β and PrP influences the dimerization of 14-3-3 and fibrillization of PrP106–126

Proteins of the 14-3-3 family are universal participate in multiple cellular processes. However, their exact role in the pathogenesis of prion diseases remains unclear. In this study, we proposed that human PrP was able to form molecular complex with 14-3-3β. The domains responsible for the interactions between PrP and 14-3-3β were mapped at the segments of amino acid (aa) residues 106–126 within PrP and aa 1–38 within 14-3-3β. Homology modeling revealed that the key aa residues for molecular interaction were D22 and D23 in 14-3-3β as well as K110 in PrP. Mutations in these aa residues inhibited the interaction between the two proteins in vitro. Our results also showed that recombinant PrP encouraged 14-3-3β dimer formation, whereas PrP106–126 peptide inhibited it. Recombinant 14-3-3β disaggregated the mature PrP106–126 fibrils in vitro. Moreover, the PrP–14-3-3 protein complexes were observed in the brain tissues of normal and scrapie agent 263 K infected hamsters. Colocalization of PrP and 14-3-3 was seen in the cytoplasm of human neuroblastoma cell line SH-SY5Y, as well as human cervical cancer cell line HeLa transiently expressing full-length human PrP. Our current data suggest the neuroprotection of PrPC and neuron damage caused by PrPSc may be associated with their functions of 14-3-3 dimerization regulation.     

PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP^C to scrapie isoform PrP^Sc). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126_CONH2 and PrP106-126_COOH) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126_COOH displayed a higher peptide-lipid binding affinity than PrP106-126_CONH2 (∼2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.     

An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds

Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.     

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.     

PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP^C, for “cellular prion protein”) into an abnormal state (PrP^Sc, for “scrapie prion protein”). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP^C. In contrast to its homologue PrP^C, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP^Sc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP^C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.     

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.     

Protective effect of edaravone against PrP106‐126–induced PC12 cell death

The prion protein peptide PrP106‐126 induces cell apoptosis through mechanisms involving production of intracellular reactive oxygen species. The present study investigated the effects of edaravone, a potent free radical scavenger in clinical use, on cell cytotoxicity induced by PrP106‐126. Results showed that PrP106‐126 decreased PC12 cell viability in a dose‐ and time‐dependent manner. Edaravone significantly antagonized the cytotoxic effects of PrP106‐126. Mechanistically, PrP106‐126 decreased PC 12 intracellular glutathione (GSH) concentrations, decreased superoxide dismutase (SOD) enzyme activity, increased concentrations of the oxidation end product malondialdehyde (MDA), depolarized the mitochondrial membrane, and increased caspase‐3 activity. Edaravone alone did not affect GSH, SOD, or MDA but did effectively reverse all of the intracellular prooxidant effects induced by PrP106‐126 and inhibit induced apoptosis in PC12 cells. In conclusion, edaravone may be a viable candidate for the treatment of oxidative stress‐induced neurodegenerative disease. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:235–241, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20330     

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.     

Beta-amyloid-derived pentapeptide RIIGLa inhibits Abeta(1-42) aggregation and toxicity

Pr-IIGL(a), a derivative of the tetrapeptide beta-amyloid 31-34 (Abeta(31-34)), exerts controversial effects: it is toxic in a neuroblastoma culture, but it protects glial cells from the cytotoxic action of Abeta(1-42). For an understanding of this phenomenon, a new pentapeptide, RIIGL(a) was synthetized, and both compounds were studied by different physicochemical and biological methods. Transmission electron microscopic (TEM) studies revealed that Pr-IIGL(a) forms fibrillar aggregates, whereas RIIGL(a) does not form fibrils. Congo red binding studies furnished the same results. Aggregated Pr-IIGL(a) acts as a cytotoxic agent in neuroblastoma cultures, but RIIGL(a) does not display inherent toxicity. RIIGL(a) co-incubated with Abeta(1-42) inhibits the formation of mature amyloid fibres (TEM studies) and reduces the cytotoxic effect of fibrillar Abeta(1-42). These results indicate that RIIGL(a) is an effective inhibitor of both the aggregation and the toxic effects of Abeta(1-42) and can serve as a lead compound for the design of novel neuroprotective peptidomimetics.     

New coumarin-benzotriazole based hybrid molecules as inhibitors of acetylcholinesterase and amyloid aggregation

A novel series of triazole tethered coumarin-benzotriazole hybrids based on donepezil skeleton has been designed and synthesized as multifunctional agents for the treatment of Alzheimer’s disease (AD). Among the synthesized compounds 13b showed most potent acetylcholinesterase (AChE) inhibition (IC₅₀ = 0.059 μΜ) with mixed type inhibition scenario. Structure-activity relationship revealed that three-carbon alkyl chain connecting coumarin and triazole is well tolerable for inhibitory potential. Hybrids obtained from 4-hydroxycoumarin and 1-benzotriazole were most potent AChE inhibitors. The inhibitory potential of all compounds against butyrylcholinesterase was also evaluated but all showed negligible activity suggesting that the hybrid molecules are selective AChE inhibitors. 13b (most potent AChE inhibitor) also showed copper-induced Aβ_1-42 aggregation inhibition (34.26% at 50 μΜ) and chelating properties for metal ions (Cu²⁺, Fe^2+, and Zn²⁺) involved in AD pathogenesis along with DNA protective potential against degenerative actions of OH radicals. Molecular modelling studies confirm the potential of 13b in blocking both PAS and CAS of AChE. In addition, interactions of 13b with Aβ_1-42 monomer are also streamlined. Therefore, hybrid 13b can act as an effective hit lead molecule for further development of selective AChE inhibitors as multifunctional anti-Alzheimer’s agents.     

Knockdown of ACAT-1 reduces amyloidogenic processing of APP

Previous studies have shown that acyl-coenzyme A:cholesterol acyl transferase (ACAT), an enzyme that controls cellular equilibrium between free cholesterol and cholesteryl esters, modulates proteolytic processing of APP in cell-based and animal models of Alzheimer's disease. Here we report that ACAT-1 RNAi reduced cellular ACAT-1 protein by approximately 50% and cholesteryl ester levels by 22% while causing a slight increase in the free cholesterol content of ER membranes. This correlated with reduced proteolytic processing of APP and 40% decrease in Abeta secretion. These data show that even a modest decrease in ACAT activity can have robust suppressive effects on Abeta generation.     

Intracellular accumulation of a mild-denatured monomer of the human PrP fragment 90-231, as possible mechanism of its neurotoxic effects

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90–231 (hPrP90–231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90–231 (incubated for 1 h at 53°C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90–231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.     

Internalization of PrP106-126 by the formyl-peptide-receptor-like-1 in glial cells

Recent studies suggest that the formyl-peptide-receptor-like-1 (FPRL1) plays an essential role in inflammatory responses in the host defence mechanisms and neurodegenerative disorders. Furthermore, it may be involved in proinflammatory processes of prion diseases. However, little is known about the induction and regulation of PrP106-126-induced receptor endocytosis. We have thus analysed whether PrP106-126 increases the activity of phospholipase D (PLD) via FPRL1, an enzyme involved in the regulation of the secretion, endocytosis and receptor signalling, in glial cells. PLD activity was determined using a transphosphatidylation assay and the internalization of PrP106-126, and FPRL1 was assessed by fluorescence microscopy and quantified by ELISA. We could show that PLD is activated by PrP106-126 both in astrocytes and microglia, and moreover that PrP106-126 is rapidly internalized via FPRL1 in astrocytes and microglia cells. The determination of receptor activity by extracellular signal-regulated kinases 1/2 phosphorylation and cAMP level measurement verified the PrP106-126-induced activation of FPRL1. FPRL1-mediated PrP106-126 uptake was blocked by the receptor antagonist chenodeoxycholic acid. These studies indicate the involvement of FPRL1-mediated cellular signalling in PrP106-126-endocytosis and may allow the development of therapeutic agents interfering with prion uptake and/or PLD function, using either PLD or the FPRL1 as a possible pharmaceutical target.     

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.