朊病毒疾病

朊病毒是一种蛋白性质的感染颗粒,它能引起动物的一类大脑功能紊乱疾病:可传染海绵样脑病(TSE)。本文就朊病毒、朊病毒引起的疾病、牛海绵样脑病(BSE)及BSE能否传给人类进行一些讨论。     

朊病毒研究的现状

朊病毒疾病是人和动物中的一种传染性,散发性和遗传性的神经退行性脑病,到目前为止,关于此病的致病机制并不十分清楚,但有研究表明该病是由一种正常蛋白PrP的不正常折叠形式在大脑中积聚所致,许多哺乳动物和鸟类的朊病毒基因和氨基酸序列都已被分析,而且传染源的部分性质已经被阐明,朊病毒疾病的传染和潜伏期在人和动物中有很大差异,一些变异与疾病的自发性,易感性和抵抗性有关,为得到关于现毒更多的信息,已对其基因两端进行了大范围的DNA测序分析,至于正常细胞蛋白PrP的生理功能,现在也并不确定。     

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。     

Remarkable increases of α1-antichymotrypsin in brain tissues of rodents during prion infection

α1-Antichymotrypsin (α1-ACT) belongs to a kind of acute-phase inflammatory protein. Recently, such protein has been proved exist in the amyloid deposits which is the hallmark of Alzheimer's disease, but limitedly reported in prion disease. To estimate the change of α1-ACT during prion infection, the levels of α1-ACT in the brain tissues of scrapie agents 263K-, 139A- and ME7-infected rodents were analyzed, respectively. Results shown that α1-ACT levels were significantly increased in the brain tissues of the three kinds of scrapie-infected rodents, displaying a time-dependent manner during prion infection. Immunohistochemistry assays revealed the increased α1-ACT mainly accumulated in some cerebral regions of rodents infected with prion, such as cortex, thalamus and cerebellum. Immunofluorescent assays illustrated ubiquitously localization of α1-ACT with GFAP positive astrocytes, Iba1-positive microglia and NeuN-positive neurons. Moreover, double-stained immunofluorescent assays and immunohistochemistry assays using series of brain slices demonstrated close morphological colocalization of α1-ACT signals with that of PrP and PrP^Sc in the brain slices of 263K-infected hamster. However, co-immunoprecipitation does not identify any detectable molecular interaction between the endogenous α1-ACT and PrP either in the brain homogenates of 263K-infected hamsters or in the lysates of prion-infected cultured cells. Our data here imply that brain α1-ACT is increased abnormally in various scrapie-infected rodent models. Direct molecular interaction between α1-ACT and PrP seems not to be essential for the morphological colocalization of those two proteins in the brain tissues of prion infection.     

Prions

Prions were originally defined as infectious agents of protein nature, which caused neurodegenerative diseases in animals and humans. The prion concept implies that the infectious agent is a protein in special conformation that can be transmitted to the normal molecules of the same protein through protein-protein interactions. Until the 1990s, the prion phenomenon was associated with the single protein named PrP. Discovery of prions in lower eukaryotes, the yeast Saccharomyces cerevisiae and fungus Podospora anserina, suggests that prions have wider significance. Prions of lower eukaryotes are not related to diseases; their propagation caused by aggregation of prion-like proteins underlies the inheritance of phenotypic traits and most likely has adaptive significance. This review covers prions of mammals and lower eukaryotes, mechanisms of their appearance de novo and maintenance, structure of prion particles, and prospects for the treatment of prion diseases. Recent data concerning the search for new prion-like proteins is included. The paper focuses on the [PSI+] prion of S. cerevisiae, since at present it is the most investigated one. The biological significance of prions is discussed.     

Recombinant neural protein PrP can bind with both recombinant and native apolipoprotein E in vitro

The most essential and crucial step during the pathogenesis of transmissible spongiformencephalopathy is the conformational change of cellular prion protein (PrP~C) to pathologic isoform (PrP~(Sc)).Alot of data revealed that caveolae-like domains (CLDs) in the cell surface were the probable place where theconversion of PrP proteins happened.Apolipoprotein E (ApoE) is an apolipoprotein which is considered toplay an important role in the development of Alzheimer's disease and other neurodegenerative diseases byforming protein complex through binding to the receptor located in the clathrin-coated pits of the cell surface.In this study,a 914-bp cDNA sequence encoding human ApoE3 was amplified from neuroblastoma cell lineSH-SY5Y.Three human ApoE isomers were expressed and purified from Escherichia coli.ApoE-specificantiserum was prepared by immunizing rabbits with the purified ApoE3.GST/His pull-down assay,immunoprecipitation and ELISA revealed that three full-length ApoE isomers interact with the recombinantfull-length PrP protein in vitro.The regions corresponding to protein binding were mapped in the N-terminalsegment of ApoE (amino acid 1-194) and the N-terminal of PrP (amino acid 23-90).Moreover,the recombinantPrP showed the ability to form a complex with the native ApoE from liver tissues.Our data provided directevidence of molecular interaction between ApoE and PrP.It also supplied scientific clues for assessing thesignificance of CLDs on the surface of cellular membrane in the process of conformational conversion fromPrP~C to PrP~(Sc) and probing into the pathogenesis of transmissible spongiform encephalopathy.     

朊粒,生命与病原体

朊粒的本质是蛋白质。但能自我复制 ,与环境进行能量交换 ,是一种耗散结构 ,从分子生物学、生物物理学以及热力学的观点来看 ,都表明朊粒具有生命现象。同时朊粒具有传染性、致病性 ,因此它能作为一种独立的病原体存在。     

Molecular Biology and Transgenetics of Prion Diseases

Abstract

Considerable progress has been made deciphering the role of an abnormal isoform of the prion protein (PrP) in scrapie of animals and Gerstmann-Sträussler syndrome (GSS) of humans. Some transgenic (Tg) mouse (Mo) lines that carry and express a Syrian hamster (Ha) PrP gene developed scrapie 75 d after inoculation with Ha prions; non-Tg mice failed to show symptoms after 500 d. Brains of these infected Tg(HaPrP) mice featured protease-resistant HaPrP^sc, amyloid plaques characteristic for Ha scrapie, and 10⁹ ID₅₀ units of Ha-specific prions upon bioassay. Studies on Syrian, Armenian, and Chinese hamsters suggest that the domain of the PrP molecule between codons 100 and 120 controls both the length of the incubation time and the deposition of PrP in amyloid plaques. Ataxic GSS in families shows genetic linkage to a mutation in the PrP gene, leading to the substitution of Leu for Pro at codon 102. Discovery of a point mutation in the Prp gene from humans with GSS established that GSS is unique among human diseases it is both genetic and infectious. These results have revised thinking about sporadic Creutzfeldt-Jakob disease, suggesting it may arise from a somatic mutation. These findings combined with those from many other studies assert that PrP^sc is a component of the transmissible particle, and the PrP amino acid sequence controls the neuropathology and species specificity of prion infectivity. The precise mechanism of PrP^& formation remains to be established. Attempts to demonstrate a scrapie-specific nucleic acid within highly purified preparations of prions have been unrewarding to date. Whether transmissible prions are composed only of PrP^sc molecules or do they also contain a second component such as small polynucleotide remains uncertain.     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic β-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. β-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated β-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded β-sheet-rich proteins accumulate.     

BSE - a wolf in sheep's clothing?

The entire sheep flock in the UK has been threatened with slaughter if BSE is found in farmed sheep, largely on the grounds that an epidemic of BSE in sheep could be harder to contain than was the case for cattle, and that lamb could present a greater risk to consumers than beef. However, identifying BSE in a sheep is not straightforward, because of its similarities to the related disease, scrapie. Here, we review the likelihood that any UK sheep have BSE, how they might have got it, how a case could be identified and what the Government is doing in terms of surveillance and possible control methods.     

Prion Protein mPrP[F175A](121-231): Structure and Stability in Solution

The three-dimensional structures of prion proteins (PrPs) in the cellular form (PrP^C) include a stacking interaction between the aromatic rings of the residues Y169 and F175, where F175 is conserved in all but two so far analyzed mammalian PrP sequences and where Y169 is strictly conserved. To investigate the structural role of F175, we characterized the variant mouse prion protein mPrP[F175A](121-231). The NMR solution structure represents a typical PrP^C-fold, and it contains a 3₁₀-helical β2-α2 loop conformation, which is well defined because all amide group signals in this loop are observed at 20 °C. With this “rigid‐loop PrP^C” behavior, mPrP[F175A](121-231) differs from the previously studied mPrP[Y169A](121-231), which contains a type I β-turn β2-α2 loop structure. When compared to other rigid‐loop variants of mPrP(121-231), mPrP[F175A](121-231) is unique in that the thermal unfolding temperature is lowered by 8 °C. These observations enable further refined dissection of the effects of different single-residue exchanges on the PrP^C conformation and their implications for the PrP^C physiological function.     

Are Brazilian cervids at risk of prion diseases?

Prion diseases are neurodegenerative fatal disorders that affect human and non-human mammals. Chronic Wasting Disease (CWD) is a prion disease of cervids regarded as a public health problem in North America, and polymorphisms at specific codons in the PRNP gene are associated with this disease. To assess the potential CWD susceptibility of South American free-ranging deer, the presence of these polymorphisms was examined in Mazama gouazoubira, Ozotoceros bezoarticus and Blastocerus dichotomus. Despite the lack of CWD reports in Brazil, the examined codons (95, 96, 116, 132, 225, and 226) of the PRNP gene showed potential CWD susceptibility in Brazilian deer. Low abundancy of deer in Brazil possibly difficult both CWD proliferation and detection, however, CWD surveillance may not be neglected.     

Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes

AIMS: To analyse the frequencies of prion (PrP) gene haplotypes in UK sheep flocks and evaluate their relevance to transmissible spongiform encephalopathies (TSEs) and TSE resistance breeding programmes in sheep. METHODS AND RESULTS: Genomic DNA isolated from sheep blood was PCR amplified for the coding region of the PrP gene and then sequenced. This study has analysed the sequence of PrP between codons 110 and 245 in 6287 ARQ haplotypes revealing a total of eight variant sequences, which represent a higher than expected 41% of all ARQ haplotypes. The additional PrP gene dimorphisms were M112T, L141F, M137T, H143R, H151C, P168L, Q175E and P241S. CONCLUSION: The results do not suggest a correlation between the occurrence of a specific ARQ haplotype and the scrapie disease status of a flock. The ARQ haplotype variability appears to be different in the UK sheep flocks compared with sheep flocks from outside the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: Additional PrP dimorphisms may impact on the methodologies used for standard PrP genotyping in sheep breeding programmes. Some of these polymorphisms were found with significant frequencies in the UK sheep flocks and should therefore be considered in breeding programmes.     

Behind the potential evolution towards prion resistant species

Historically, the observation of naturally occurring cases of prion disease led to the classification of different susceptibility grades and to the designation of prion resistant species. However, the development of highly efficient in vitro prion propagation systems and the generation of ad hoc transgenic models allowed determining that leporidae and equidae families have been erroneously considered resistant to prion infection. On the contrary, similar approaches revealed an unexpected high level of resistance of the canidae family. In PLoS Pathogens [1 Fernandez-Borges N, Parra B, Vidal E, et al. Unraveling the key to the resistance of canids to prion diseases. PLoS Pathog. 2017;13:e1006716. doi:10.1371/journal.ppat.1006716. eCollection 2017 Nov. PMID: 29131852[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]], we describe experiments directed toward elucidating which are the determinants of the alleged prion resistance of this family. Studies based on the sequence of the canine prion protein coupled with structural in silico analysis identified a key residue probably implicated in this resistance. Cell and brain-based PMCA highlighted that the presence of aspartic or glutamic acid at codon 163 of the canid PrP, strongly inhibits prion replication in vitro. Transgenic animals carrying this substitution in mouse PrP were resistant to prion infection after intracerebral challenge with different mouse prion strains. The confirmation of the importance of this substitution and its exclusivity in this family, suggests it could have been evolutionarily favored, due to their diet based on carrion and small ruminants.     

蛋白质的错误折叠与疾病

蛋白质是生物体内一切功能的执行者.人体内的任何功能,从催化化学反应到抵御外来侵略都是蛋白质作用的结果.蛋白质折叠是生命活动的最基本过程,近年发现蛋白质的错误折叠可以导致一些疾病.蛋白质的错误折叠与疾病的关系已成为分子生物学新的研究前沿.介绍了细胞内保证蛋白质正常功能的“质量控制”系统,重点讨论了翻译后的质量控制、与蛋白质错误折叠有关的一些疾病和治疗这一类疾病的原则方法.