Toward the molecular basis of inherited prion diseases: NMR structure of the human prion protein with V210I mutation

The development of transmissible spongiform encephalopathies (TSEs) is associated with the conversion of the cellular prion protein (PrP^C) into a misfolded, pathogenic isoform (PrP^Sc). Spontaneous generation of PrP^Sc in inherited forms of disease is caused by mutations in gene coding for PrP (PRNP). In this work, we describe the NMR solution-state structure of the truncated recombinant human PrP (HuPrP) carrying the pathological V210I mutation linked to genetic Creutzfeldt-Jakob disease. The three-dimensional structure of V210I mutant consists of an unstructured N-terminal part (residues 90-124) and a well-defined C-terminal domain (residues 125-228). The C-terminal domain contains three α-helices (residues 144-156, 170-194 and 200-228) and a short antiparallel β-sheet (residues 129-130 and 162-163). Comparison with the structure of the wild-type HuPrP revealed that although two structures share similar global architecture, mutation introduces some local structural differences. The observed variations are mostly clustered in the α₂-α₃ inter-helical interface and in the β₂-α₂ loop region. Introduction of bulkier Ile at position 210 induces reorientations of several residues that are part of hydrophobic core, thus influencing α₂-α₃ inter-helical interactions. Another important structural feature involves the alteration of conformation of the β₂-α₂ loop region and the subsequent exposure of hydrophobic cluster to solvent, which facilitates intermolecular interactions involved in spontaneous generation of PrP^Sc. The NMR structure of V210I mutant offers new clues about the earliest events of the pathogenic conversion process that could be used for the development of antiprion drugs.     

Prion protein glycosylation

The transmissible spongiform encephalopathies (TSE), or prion diseases are a group of transmissible neurodegenerative disorders of humans and animals. Although the infectious agent (the 'prion') has not yet been formally defined at the molecular level, much evidence exists to suggest that the major or sole component is an abnormal isoform of the host encoded prion protein (PrP). Different strains or isolates of the infectious agent exist, which exhibit characteristic disease phenotypes when transmitted to susceptible animals. In the absence of a nucleic acid genome it has been hard to accommodate the existence of TSE strains within the protein-only model of prion replication. Recent work examining the conformation and glycosylation patterns of disease-associated PrP has shown that these post-translational modifications show strain-specific properties and contribute to the molecular basis of TSE strain variation. This article will review the role of glycosylation in the susceptibility of cellular PrP to conversion to the disease-associated conformation and the role of glycosylation as a marker of TSE strain type.     

Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases

Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrP^Sc in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrP^Sc in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrP^Sc in a prion-infected cell line after treatment with bafilomycin A₁. These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.     

Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases

Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrP^Sc in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrP^Sc in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrP^Sc in a prion-infected cell line after treatment with bafilomycin A₁. These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.     

单核苷酸多态概述

单核苷酸多态ＳＮＰ是遍布于基因组中的一种ＤＮＡ序列变化类型,人类基因组中平均约每一千碱基中有一个。单核苷酸多态是一种双等位型多态,群体中出现的频率大于１％或２％者视为多态,低于１％或２％的则视为突变。由于其具有高信息量、高密度又便于自动化操作的特点,单核苷酸多态在遗传性疾病基因的克隆和药物的设计与开发方面具有广阔的应用前景。本文对单核苷酸的概念、特点、应用前景,及其研究应用的一些问题作一综述。     

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.     

传染性海绵样脑病病原的研究进展

传染性海绵样脑病因疯牛病的爆发和新型克雅氏病的出现而引起人们的关注。研究发现,疯牛病的流行可能是由羊痒病的病原引起的,这病原被Ｐｒｕｓｉｎｅｒ命名为“Ｐｒｉｏｎ”（１９８２）。从８０年代开始研究以来,越来越多的关于Ｐｒｉｏｎ的特性被人们所了解,比如Ｐｒｉｏｎ蛋白和ＰｒＰ基因。本文综述了近几年来研究者们用不同手段从各个角度对病原进行研究的新进展。     

单核苷酸多态性基因分型技术原理与进展

在基因组规模了解遗传变异与生物功能之间的关系可望为生物学带来全新的深入认识。本从等位基因分型机理、反应形式和检测方法等三个方面讨论SNP分型方法的现状,并简要介绍了目前应用的一些分型方法。     

Review. The epidemiology of kuru: monitoring the epidemic from its peak to its end

Kuru is a fatal transmissible spongiform encephalopathy restricted to the Fore people and their neighbours in a remote region of the Eastern Highlands of Papua New Guinea. When first investigated in 1957 it was found to be present in epidemic proportions, with approximately 1000 deaths in the first 5 years, 1957-1961. The changing epidemiological patterns and other significant findings such as the transmissibility of kuru are described in their historical progression. Monitoring the progress of the epidemic has been carried out by epidemiological surveillance in the field for 50 years. From its peak, the number of deaths from kuru declined to 2 in the last 5 years, indicating that the epidemic is approaching its end. The mode of transmission of the prion agent of kuru was the local mortuary practice of transumption. The prohibition of this practice in the 1950s led to the decline in the epidemic, which has been prolonged into the present century by incubation periods that may exceed 50 years. Currently, the epidemiological surveillance is being maintained and further studies on human genetics and the past mortuary practices are being conducted in the kuru-affected region and in communities beyond it.     

羊瘙痒因子263K毒株感染金黄地鼠后鼠脑组织中检出PrP-res蛋白及其神经病理学研究

羊瘙痒病是累及山羊及绵羊的可传播海绵状脑病。为了观察羊瘙痒因子 (Scrapie)的病原特征及病理组织改变特点 ,将羊瘙痒因子 2 6 3K毒株颅内接种至金黄地鼠。经过 81～ 110天的潜伏期 ,89%的动物发病 (17/19只 )。对发病地鼠的神经病理学检测发现 ,海绵状空泡变性的检出率为 5 9% ,淀粉样斑的检出率为 17 6 %。利用免疫组化和蛋白酶消化后的Westernblotting检测证实 ,10 0 %的发病地鼠的脑组织中都出现蛋白酶抗性朊蛋白 (PrP res)。17只发病地鼠脑组织提取物中 ,PrP res的泳动位置和分子量大小完全一致 ,出现两条分子量在 2 5kD～ 31kD的反应带。尝试应用快速玻片印迹法检测病变组织中的PrP res,结果显示 ,与常规固定包埋切片的免疫组化检出效果相似。这提示脑组织印片法可成为临床检测克 雅氏病 (Creutzfeldt Jacobdisease ,CJD)患者脑组织活检标本中PrP res的快速、有效的方法。羊瘙痒因子 2 6 3K成功感染金黄地鼠再次证明 ,金黄地鼠是TSE感染因子良好的动物模型 ,发病率高 ,潜伏期短 ,发病动物PrP res的检出率明显高于典型病理改变的检出率。新生成的PrP res的电泳类型与接种的TSE因子有关 ,与宿主的个体差异无关 ,提示TSE感染因子的确存在“株”的现象。     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.