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.     

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。     

Prion protein fragment 106-126 potentiates catecholamine secretion from PC-12 cells

The toxic actions of scrapie prion protein(PrP^sc) are poorly understood. We investigated the abilityof the toxic PrP^sc fragment 106-126 to interfere withevoked catecholamine secretion from PC-12 cells. Prion protein fragment106-126 (PrP106-126) caused a time- andconcentration-dependent augmentation of exocytosis due to the emergenceof a Ca²⁺ influx pathway resistant to Cd²⁺ butsensitive to other inorganic cations. In control cells, secretion wasdependent on Ca²⁺ influx through L- and N-typeCa²⁺ channels, but after exposure to PrP106-126,secretion was unaffected by N-type channel blockade. Instead, selectiveL-type channel blockade was as effective as Cd²⁺ insuppressing secretion. Patch-clamp recordings revealed no change intotal Ca²⁺ current density in PrP106-126-treated cellsor in the contribution to total current of L-type channels, but a smallCd²⁺-resistant current was found only inPrP106-126-treated cells. Thus PrP106-126 augments secretionby inducing a Cd²⁺-resistant Ca²⁺ influxpathway and alters coupling of native Ca²⁺ channels toexocytosis. These effects are likely contributory factors in the toxiccellular actions of PrP^sc.

     

Reversible Effects of Peptide Concentration and Lipid Composition on H-Ras Lipid Anchor Clustering

Dynamic clusters of lipid-anchored Ras proteins are important for high-fidelity signal transduction in cells. The average size of Ras nanoclusters was reported to be independent of protein expression levels, and cholesterol depletion is commonly used to test the raft-preference of nanoclusters. However, whether protein concentration and membrane domain stability affect Ras clustering in a reversible manner is not well understood. We used coarse-grained molecular dynamics simulations to examine the reversibility of the effects of peptide and cholesterol concentrations as well as a lipid domain-perturbing nanoparticle (C₆₀) on the dynamics and stability of H-Ras lipid-anchor nanoclusters. By comparing results from these simulations with previous observations from the literature, we show that effects of peptide/cholesterol concentrations on the dynamics and stability of H-Ras peptide nanoclusters are reversible. Our results also suggest a correlation between the stabilities of lipid domains and Ras nanoclusters, which is supported by our finding that C₆₀ penetrates into the liquid-disordered domain of the bilayer, destabilizing lipid domains and thereby the stability of the nanoclusters.     

Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel

Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding the many cellular processes. Recently, we have found that dimeric αβ-tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of mitochondrial outer membrane. Here, we show that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude.     

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.     

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.     

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.     

The Effect of Membrane Lipid Composition on the Formation of Lipid Ultrananodomains

Some lipid mixtures form membranes containing submicroscopic (nanodomain) ordered lipid domains (rafts). Some of these nanodomains are so small (radius <5 nm) that they cannot be readily detected with Förster resonance energy transfer (FRET)-labeled lipid pairs with large Ro. We define such domains as ultrananodomains. We studied the effect of lipid structure/composition on the formation of ultrananodomains in lipid vesicles using a dual-FRET-pair approach in which only one FRET pair had Ro values that were sufficiently small to detect the ultrananodomains. Using this approach, we measured the temperature dependence of domain and ultrananodomain formation for vesicles composed of various mixtures containing a high-T_m lipid (brain sphingomyelin (SM)) or dipalmitoyl phosphatidylcholine (DPPC)), low-T_m lipid (dioleoylphosphatidylcholine (DOPC) or 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC)), and a lower (28 mol %) or higher (38 mol %) cholesterol concentration. For every lipid combination tested, the thermal stabilities of the ordered domains were similar, in agreement with our prior studies. However, the range of temperatures over which ultrananodomains formed was highly lipid-type dependent. Overall, vesicles that were closest to mammalian plasma membrane in lipid composition (i.e., with brain SM, POPC, and/or higher cholesterol) formed ultrananodomains in preference to larger domains over the widest temperature range. Relative to DPPC, the favorable effect of SM on ultrananodomain formation versus larger domains was especially large. In addition, the favorable effect of a high cholesterol concentration, and of POPC versus DOPC, on the formation of ultrananodomains versus larger domains was greater in vesicles containing SM than in those containing DPPC. We speculate that it is likely that natural mammalian lipids are tuned to maximize the tendency to form ultrananodomains relative to larger domains. The observation that domain size is more sensitive than domain formation to membrane composition has implications for how membrane domain properties may be regulated in vivo.     

Heat shock protein 104 inhibited the fibrillization of prion peptide 106-126 and disassembled prion peptide 106-126 fibrils in vitro

Amyloid-like fibrils have been associated with the pathogenesis of human prion diseases. Prion peptide of aa 106-126 (PrP106-126) exhibits many PrP(Sc)-like biochemical features, forming amyloid-like fibrils in vitro. Here, we found that the recombinant yeast-derived molecular chaperon Hsp104 inhibited significantly the fibril assembly of the synthetic PrP106-126 peptide by dynamic ThT assays in vitro. EM assays revealed almost no fibril-like structure after incubation of the synthetic PrP106-126 peptides with Hsp104 for 12h. Circular dichroism assays identified that treatment of Hsp104 shifted the secondary structure of PrP106-126 fibrils from β-sheet to a random coil. MTT tests confirmed that interaction of PrP106-126 with Hsp104 maintained the toxicity of PrP106-126 on human neuroblastoma cell line SK-N-SH. Additionally, Hsp104 was able to disassemble the mature PrP106-126 fibrils in vitro, leading to recovering the cytotoxicity of PrP106-126 on SK-N-SH cells. Our study provides the molecular evidences that the yeast-derived Hsp104 can interfere in the fibril assembly and disassembly of human PrP106-126 segment.     

RFP134对UMR106细胞膜脂流动性的影响(英文)

癌细胞具有与正常细胞不同的膜脂流动性,导致细胞对生长因子和癌基因产物反应敏感;引起细胞增殖失控。本实验室从植物中发现一种二萜类活性物质──RFP134,在细胞周期和信号传递等多方面表现出有抑制癌细胞增殖,促进细胞分化的作用。本文以大鼠成骨肉瘤细胞（UMR106）和正常大鼠成骨细胞为模型,研究其对癌细胞膜脂流动性的影响。细胞系UMR106由美国麻省总医院内分泌室赠送。成骨细胞由本实验室分离培养。以不同浓度（20、40、60、80、100μM／L）的RFP134,在同一时间处理细胞,或以最适浓度（50μM／L）在不同时间作用于细胞。DPH为荧光标记物,测得的荧光偏振值和微粘度值为膜膜流动性指标。结果显示,无论在恒定的时间、以不同浓度的RFP134作用于UMR106细胞（Fig.1B）,或以恒定的浓度、在不同时间处理UMR106细胞（Fig．1D）,结果均表现为显著降低膜脂流动性。前者,RFP134作用于细胞时,细胞荧光偏振值与微粘度值逐步升高,其变化呈量效关系;而后者,呈时效关系。但在最适浓度与最佳作用时间,荧光偏振值和微粘度值达饱和状态。在同样条件下,RFP134对正常成骨细胞的膜脂流动性影响极小。即：荧光偏振值和微粘度值均在正常范围内保持恒定（Fig．1A;Fig．1C）。RFP134降低癌细胞的膜脂流动性     

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.     

Contrasting Effects of Two Lipid Cofactors of Prion Replication on the Conformation of the Prion Protein

Recent studies introduced two experimental protocols for converting full-length recombinant prion protein (rPrP) purified from E.coli into the infectious prion state (PrP^Sc) with high infectivity titers. Both protocols employed protein misfolding cyclic amplification (PMCA) for generating PrP^Scde novo, but used two different lipids, 1-palmitoyl-2-oleolyl-sn-glycero-3-phospho(1’-rac-glycerol) (POPG) or phosphatidylethanolamine (PE), as conversion cofactors. The current study compares the effect of POPG and PE on the physical properties of native, α-helical full-length mouse rPrP under the solvent conditions used for converting rPrP into PrP^Sc. Surprisingly, the effects of POPG and PE on rPrP physical properties, including its conformation, thermodynamic stability, aggregation state and interaction with a lipid, were found to be remarkably different. PE was shown to have minimal, if any, effects on rPrP thermodynamic stability, cooperativity of unfolding, immediate solvent environment or aggregation state. In fact, little evidence indicates that PE interacts with rPrP directly. In contrast, POPG was found to bind to and induce dramatic changes in rPrP structure, including a loss of α-helical conformation and formation of large lipid-protein aggregates that were resistant to partially denaturing conditions. These results suggest that the mechanisms by which lipids assist conversion of rPrP into PrP^Sc might be fundamentally different for POPG and PE.     

Lipid Headgroups Modulate Membrane Insertion of pHLIP Peptide

The pH low insertion peptide (pHLIP) is an important tool for drug delivery and visualization of acidic tissues produced by various maladies, including cancer, inflammation, and ischemia. Numerous studies indicate that pHLIP exists in three states: unfolded and soluble in water at neutral pH (State I), unfolded and bound to the surface of a phosphatidylcholine membrane at neutral pH (State II), and inserted across the membrane as an α-helix at low pH (State III). Here we report how changes in lipid composition modulate this insertion scheme. First, the presence of either anionic lipids, cholesterol, or phosphoethanolamine eliminates membrane binding at neutral pH (State II). Second, the apparent pK_a for the insertion transition (State I → State III) is increased with increasing content of anionic lipids, suggesting that electrostatic interactions in the interfacial region modulate protonation of acidic residues of pHLIP responsible for transbilayer insertion. These findings indicate a possibility for triggering protonation-coupled conformational switching in proteins at membrane interfaces through changes in lipid composition.     

Effects of Culture Method and Growth Phase on Free Lipid Composition of Yersinia pseudotuberculosis

The influence of culture method (free-floating cells in liquid nutrient broth or bacteria attached to agar surface on solid agarized medium of the same formulation) and bacterial age on the composition of free lipids in Yersinia pseudotuber-culosis (O:Ib serovar, strain KS 3058) grown in the cold (5°C) has been investigated. The specific growth rate of the bacteria on solid medium was about threefold less than that in liquid medium. The qualitative composition of phospholipids and fatty acids only slightly depended on the bacterial culture method. At the same time, the colonially growing cultures contained somewhat more total lipids, they synthesized more phospholipids, in the linear growth phase they contained more lysophosphatides, and they had higher fatty acid unsaturation index and higher pathogenic potential than their planktonic counterparts grown in otherwise identical conditions. The bacterial growth phase influenced the amount of 3-hydroxytetrade-canoic acid and, indirectly, that of lipopolysaccharide. The dynamics of changes in the amount of this acid with bacterial age was opposite in the surface and broth cultures.     

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.     

Prion protein fragment PrP-(106-126) induces apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells

The synthetic peptide PrP-(106-126) has previously been shown to be neurotoxic. Here, for the first time, we report that it induces apoptosis in the human neuroblastoma cell line SH-SY5Y. The earliest detectable apoptotic event in this system is the rapid depolarization of mitochondrial membranes, occurring immediately upon treatment of cells with PrP-(106-126). Subsequent to this, cytochrome c release and caspase activation were observed. Caspase inhibitors demonstrated that while the peptide activates caspases they are not an absolute requirement for apoptosis. Parallel to caspase activation, PrP-(106-126) was also observed to trigger a rise in intracellular calcium through release of mitochondrial calcium stores. This leads to the activation of calpains, another family of proteases. A calpain inhibitor demonstrated that while calpains are activated by the peptide they also are not an absolute requirement for apoptosis. Interestingly a combination of caspase and calpain inhibitors significantly inhibited apoptosis. This illustrates alternative pathways leading to apoptosis via caspases and calpains and that blocking both pathways is required to inhibit apoptosis. These results implicate the mitochondrion as a primary site of action of PrP-(106-126).     

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP^C into pathogenic PrP^Sc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.