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.     

两种PrP截短型多肽的神经毒性

PrP^106-126和PrP^118-135分别对应于人PrP^c序列中106～126残基和118～135残基,两个肽段在试验中具备prpsc的部分病理特症,因此PrP^106-126和PrP^118-135就成为了研究PrP^Sc神经细胞毒性致病机制的合适的模型。     

PrP106-126多肽诱导凋亡细胞中14-3-3β的降解

本研究将PrP106-126多肽和HeLa细胞共孵育4h和8h,采用Hoechst染色分析发现PrP106-126诱导凋亡细胞细胞核出现不同程度的染色质浓集,固缩及碎裂的细胞凋亡征象。Western blotting检测发现PrP106-126诱导细胞中的多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)降解,提示PrP106-126通过caspase3途径引起细胞凋亡现象。PrP106-126诱导的细胞中14-3-3β在不同孵育时间也出现降解,而Real-time PCR检测14-3-3β mRNA未发生变化。本研究证明PrP106-126通过caspase3诱导HeLa细胞凋亡,并可导致抗凋亡蛋白14-3-3β的降解而加速凋亡的形成。     

全长朊粒蛋白淀粉样纤维引发细胞毒性的机制研究

目的 朊病毒病(prion disease)是一类由朊粒蛋白(PrP)发生错误折叠、聚集形成致病性的PrP^Sc导致的具有高致死率的神经退行性疾病。本文在细胞和动物水平开展了PrP纤维诱导内源PrP聚集和毒性机制的研究。方法 通过超速离心结合蛋白质免疫印迹实验检测PrP聚集；通过氧化压力实验，使用Annexin V-FITC/PI双染检测细胞凋亡；运用细胞超薄切片技术检测细胞线粒体形态；在动物水平，分离新生小鼠的前额叶，进行横断切片培养，在脑片上接种PrP纤维。结果 PrP纤维种子可以诱导内源PrP聚集，PrP纤维可以诱导细胞内氧化压力升高和细胞凋亡，PrP纤维可以引起线粒体损伤，PrP纤维可以诱导小鼠前额叶内源PrP聚集。结论 本文在细胞和动物水平证实体外组装的PrP淀粉样纤维具有细胞毒性和潜在的感染性。     

朊蛋白与信号蛋白14-3-3β的相互作用研究

为进一步确定PrP蛋白与14-3-3蛋白是否发生分子间的相互作用并确定PrP蛋白与14-3-3蛋白相互作用的区域,利用免疫共沉淀、pull down和能量共振转移(FRET)实验检测PrP蛋白与人14-3-3蛋白是否发生分子间的相互作用及相互作用的部位。结果证明,PrP蛋白与人14-3-3蛋白在体外、组织水平及细胞水平均可以发生相互作用,且证实作用的部位在PrP蛋白的106-126位氨基酸。该结果为进一步研究14-3-3蛋白在Prion疾病中的影响及Prion疾病的发病机制奠定了一定基础。     

硫醇基团的氧化还原过程对人正常朊蛋白聚集和纤维化特征的影响

本研究旨在通过体外对纯化的原核重组人正常朊蛋白硫醇基团的氧化还原过程,探究二硫键的改变对其生化特性的影响。蛋白沉淀实验显示重组正常人朊病毒蛋白经过硫醇基团的氧化还原过程明显增加了其聚集性;硫磺素T(Thioflavin T ,ThT)实验测定发现,经过硫醇基团的氧化还原过程重组PrP蛋白的纤维形成增多;圆二色谱（Circular Dichroism, CD）测定显示,处理后的重组PrP蛋白二级结构发生改变,其β-折叠结构比例显著增多;蛋白酶K消化实验也进一步显示硫醇基团的氧化还原后PrP的蛋白酶K抵抗能力有所增加。这些结果提示二硫键的形成可明显地改变PrP的二级结构,促进朊蛋白聚集和成纤维过程。     

不同缺氧时间对神经细胞中外源性HSP70表达的影响

目的:探讨不同缺氧时间刺激对神经细胞中外源性HSP70基因表达的影响.方法:采用重组腺病毒vAd-HSP70感染体外原代培养的神经细胞,48h后给予感染细胞不同缺氧时间(缺氧0h,0.5h,1h,2h,3h,4h)刺激,再复氧处理后,用RT-PCR和Westernblotting检测重组腺病毒介导的HSP70基因在神经元和胶质细胞中的转录表达.结果:重组腺病毒vAd-HSP70感染的神经细胞可检测到外源性HSP70基因的转录表达.经缺氧再复氧处理后,随着缺氧时间延长,HSP70转录水平和蛋白表达都增加,缺氧1h时最高(1.1539±0.0315,0.9699±0.0023),其次是缺氧2h时(1.0398±0.0723,0.9622±0.0026),随后依次降低,缺氧4h组(0.7477±0.0328,0.9335±0.0034)HSP70表达最低,与其他各组比较有统计学意义(P<0.05).结论:缺氧2h内给予神经细胞再复氧处理外源性HSP70表达水平较高,有利于神经细胞功能的恢复.     

重组质粒二级结构对丙型肝炎病毒E2蛋白基因表达的影响

将含HCV/E2基因的重组质粒 pUC18/HCV -E2的BamHI酶切基因片段和NcoⅠ +HindⅢ酶切基因片段分别克隆到原核表达载体pRSETHis中 ,构建两株重组HCV/E2重组表达质粒 pRSET·C/E2和 pRSET·A/E2。在诱导表达中 pRSET·A/E2的外源基因获得了高效表达 ,而 pRSET·C/E2的外源基因完全未得到表达。     

乳珍对不同给氧条件下大脑神经细胞培养的影响

已有报道,大鼠服用牛初乳制品“乳珍”后,迷宫学习训练次数减少;也有实验表明,服用后,人鼠耐受缺氧的能力得到增强。本研究以体外培养的方法,观察乳珍是否能促进大脑神经细胞的发育并提高神经细胞耐受缺氧的能力。 1 材料和方法 1.1 神经细胞的制备和给氧条件的控制 本实验采用的细胞分离和培养方法已有描述。取8日龄来亨鸡胚前脑制备成细胞悬液,然后接种于预先涂以多聚赖氨酸(Sigma)的24孔培养板(Costar)孔内,4     

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.     

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.     

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.     

Prion Protein Fragment 106–126 Differentially Induces Heme Oxygenase-1 mRNA in Cultured Neurons and Astroglial Cells

Abstract: Heme oxygenase (HO), which catalyzes the degradation of heme, has two isozymes (HO-1 and HO-2). In brain the noninducible HO-2 isoform is predominant, whereas the inducible HO-1 is a marker of oxidative stress. Because brain oxidative stress might be present in prion-related encephalopathies (PREs), as in other neurodegenerative diseases, we investigated whether HO-1 mRNA was induced in neuronal and astroglial cell cultures by a peptide corresponding to residue 106–126 of human prion protein (PrP). This peptide is amyloidogenic, and when added in vitro to cultured cells it reproduces the neuronal death and astroglial proliferation and hypertrophy occurring in PREs. HO-1 mRNA did not accumulate in rat cultured neurons from hippocampus or cortex exposed to PrP 106–126 (50 µ M for 5 days). PrP 106–126 induced HO-1 mRNA accumulation in rat astroglial cultures depending on the exposure time and concentration, being maximal (33-fold) after 7 days of exposure at 50 µ M . The nonamyloidogenic amidated or amidated-acetylated PrP 106–126 was ineffective, as was a scrambled peptide used as control. N -Acetylcysteine reduced (50%) the accumulation of HO-1 mRNA in astroglial cells after PrP 106–126 (25 µ M ) given for 5 days. Thus, oxidative stress is apparently a feature of the toxicity of PrP 106–126, and it might also occur in PREs; induction of HO-1 could contribute to the greater resistance of astrocytes compared with neurons to PrP 106–126 toxicity.     

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.     

金黄地鼠PrP基因组织特异性表达的研究

金黄地鼠是研究动物传染性海绵状脑病的理想模型动物之一,我们利用实时荧光定量RT-PCR技术,构建标准重组质粒制备标准曲线,对中枢神经系统的4个不同部位及外周6个组织提取总RNA,反转录后进行PrP基因表达的定量.结果发现,脑的四个检测部位都呈现高的表达量;在外周器官中,淋巴结的表达量和全脑相当,脾脏、心脏、肝脏和肺脏呈现中等程度的表达,肾脏的表达量最低.本研究的结果对于探讨朊蛋白的基本功能和不同组织在传染性海绵状病理发生中的作用,提供了基础数据.     

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.     

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.     

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.