朊蛋白构象改变和种属突破机制的研究进展

异常朊蛋白(P rP sc)是一种可引起人和动物传染性脑退化病的不含核酸的蛋白因子。P rP c是一个正常的蛋白,有正常的生理功能,主要分布于神经元表面,属于肌醇磷脂锚蛋白类。由P rP c向P rP sc转变即产生疾病。作者简单介绍了正常和异常朊蛋白结构、功能、构象改变及朊病毒种属屏障的研究进展,并提出了目前尚待研究的问题。     

感染性朊蛋白的毒性与糖磷脂酰肌醇（GPI）的相关性

海绵状脑病(朊病)是一类神经退行性脑病,目前是世界上研究的热点问题之一。引起该病的病原是一种朊蛋白质,它与宿主自身的正常朊蛋白的一级结构相同,只是二级结构的构象有所不同。对海绵状脑病的致病机理至今仍不太清楚,作者针对目前研究的朊蛋白的致病性与GPI的关系作一综述。     

蛋白粒子的全新传染机制

蛋白粒子主要引起人和动物的中枢神经系统退行性疾病,称之为蛋白粒子病。许多研究表明蛋白粒子病的致病因子缺乏棱酸,而是由特异的传染性蛋白质组成。该蛋白粒子是一种膜糖蛋白,至少有两种基本形式,即PrP~c和PrP~(ac)。PrP~(ac)是发病的直接原因,而PrP~c向PrP~(ac)转变则是发病的必要条件。文章就蛋白粒子概念、分子生物学以及全新的传染机制作了综合论述。     

绵羊重组朊蛋白特异性抗体的制备

传染性海绵状脑病(transmissible spongiform encephalopathy,TSE)是一类累及人和动物的中枢神经系统的退行性、致死性疾病[1],其病因目前认为是神经细胞表面的一种正常朊蛋白PrPC构象发生改变形成异常朊蛋白PrPSc所致.     

朊病毒致病机理的研究进展

就细胞型朊蛋白PrP^c的分子生物学特性、朊蛋白的生理功能、致病性朊蛋白PrP^sc对免疫系统的影响、朊病毒的外周致病机理以及相关细胞因子、化学因子与朊病毒神经侵袭之间的关系、朊病毒从外周到中枢的转运机制、入侵门户、血液传播及朊病的其他病理标志和检测方法的研究做一综述。     

不同物种SPRN基因编码区生物信息学分析

对来自26个物种的70条SPRN CDS序列及其相应氨基酸序列进行了生物信息分析.结果表明:检测到的229个多态位点中.其中有26个单一变异位点,203个简约变异位点.一个高度保守的N-端信号序列,精氨酸丰富的基本区域含有多达6个XXRG(其中X是G,A或S)重复,20个残基疏水区域具有较强的朊蛋白同源性.     

朊蛋白基因家族在畜牧兽医中的研究进展

朊蛋白基因PRNP作为朊蛋白基因家族的一员,其编码的异常朊蛋白(prion)导致传染性海绵样脑病(TSEs)的发生。PRNP基因在定位、结构、表达和编码产物上与朊蛋白基因家族其他3个基因(PRND,PRNT和SPRN)存在很多相似之处,且该基因家族在不同种属之间十分保守,朊蛋白基因家族因此被称为"朊蛋白基因复合体"(prion gene complex)。已有资料表明,朊蛋白基因家族除了与TSEs的发生密切相关,其还对畜牧生产和兽医预防有着重要影响。论文介绍了朊蛋白基因家族的4个基因及其编码产物,而后依次阐述了该基因家族对畜牧生产的影响以及在兽医预防中的研究进展,以期为畜牧生产和兽医预防提供参考。     

朊蛋白生理功能的研究进展

朊蛋白（prion protein,PrPC）作为朊病的主要相关致病因子而受到广泛关注,虽然PrPC在疾病过程中的作用已有很多了解,但关于PrPC的正常生理功能还不是很清楚。就目前的研究而言,PrPC存在细胞核亚型和细胞质亚型两种形式,可与相应的配体及铜离子作用,并且通过细胞内吞或内陷方式被细胞膜获取;主要生理功能表现为保护神经系统,协助细胞的凋亡和分化、黏附,以及参与信号转导等。因此,文章对朊蛋白的生理功能作一总结,为朊病的治疗和朊蛋白的进一步研究提供了理论基础。     

朊蛋白在宁夏滩羊不同组织中表达的免疫组化研究

作者运用免疫组织化学技术,首次对我国一类保护物种宁夏滩羊各组织中朊蛋白(PrP)的分布进行定性、定位研究。结果显示,PrP在滩羊的大脑、脑干、小脑、心脏、肝脏、脾脏、肾脏及淋巴结均有表达,尤其在脾脏、肾脏、脑干、淋巴结中的表达量丰富,而肺脏检测结果为阴性。此研究确定了朊蛋白在滩羊各组织中的表达与分布状况,并优化了朊蛋白检测的技术条件,为羊痒病的诊断与检测提供技术支持。     

PrP蛋白与Shadoo蛋白研究进展

朊蛋白(PrP蛋白)在传染性海绵状脑病中具有重要作用,但其生物学功能至今没有明确。Shadoo蛋白是一种与朊蛋白在N端结构上极为相似的新蛋白,SPRN是Shadoo蛋白的结构基因。由于Shadoo蛋白与PrP蛋白具有相同的生物学性质,且它们在脑组织中重叠表达,因此也被认为是研究朊蛋白的关键因素。文章主要探讨PrP蛋白与Shadoo蛋白在基因结构、蛋白功能、朊病毒感染方面的关系。     

PrP genetics in ruminant transmissible spongiform encephalopathies

Scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) are prion diseases in ruminants with considerable impact on animal health and welfare. They can also pose a risk to human health and control is therefore an important issue. Prion protein (PrP) genetics may be used to control and eventually eradicate animal prion diseases. The PrP gene in sheep and other representatives of the order Artiodactyles has many polymorphisms of which several are crucial determinants of susceptibility to prion diseases, also known as transmissible spongiform encephalopathies (TSE). This review will present the current understanding of PrP genetics in ruminants highlighting similarity and difference between the species in the context of TSE.     

实时荧光定量PCR检测PrP基因在金黄地鼠脑组织的动态表达

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,其脑组织内朊蛋白基因动态表达数据的测定对探讨该类疾病的发生、发展和分子致病机理具有重要意义。我们利用实时荧光定量RT-PCR技术,对不同年龄金黄地鼠大脑、小脑、丘脑和脑干PrP基因的表达进行了定量。结果发现,脑的四个检测部位都呈现高的表达量,但是同一年龄段不同组织每纳克总RNA中朊蛋白基因的表达量和每毫克组织中朊蛋白基因的表达量有显著的差别,不同组织在不同年龄出现表达高峰。本研究的结果对于探讨朊蛋白的基本功能和脑组织在传染性海绵状脑病病理发生中的作用,提供了基础数据。     

Prion agent diversity and species barrier

Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.     

Failure to detect prion protein (PrPres) by immunohistochemistry in striated muscle tissues of animals experimentally inoculated with agents of transmissible spongiform encephalopathy

Transmissible spongiform encephalopathies (TSEs) are fatal neurologic diseases. Infection by the causative agent, a prion, induces accumulations of an abnormal form of prion protein (PrP(res)) in tissues of nervous and lymphoid systems. Presence of characteristic histopathologic changes (spongiform encephalopathy) and detection of protease-resistant PrP(res) in neural and lymphoid tissues are the basis of currently available methods for diagnosis of TSEs. In this study, samples of striated muscle tissues (tongue, heart, diaphragm, and masseter muscle) from 20 animals (cattle, sheep, elk, and raccoons) were examined for PrP(res) by immunohistochemistry (IHC). All the animals had developed a TSE after experimental inoculation. PrP(res) was found by IHC in the brain but not in the muscle tissues of all the animals examined. These findings are contradictory to recently published reports of laboratory animals with TSEs, where these altered prion proteins were detected in tongue and other striated muscles. Further testing of muscle tissues is needed to confirm the findings of the present study.     

Therapeutic approaches targeting the prion receptor LRP/LR

Transmissible spongiform encephalopathies known as prion diseases are a group of fatal neurodegenerative disorders that affect both humans and animals. The generally accepted principle of the disease is that the conversion of the cellular prion protein (PrP(c)) into the disease associated isoform PrP(Sc) leads to spongiform degeneration of the brain and amyloid plaque formation. Until now no therapy leading to potential alleviation or even cure of the disease exists. It is therefore important to develop therapeutic approaches for the treatment of TSEs since these infections are inevitably fatal and, especially in the case of vCJD, they affect youngsters. Besides current conventional therapeutic strategies, this review summarizes new therapeutic tools targeting the prion receptor LRP/LR.     

Rodent models for prion diseases

Until today most prion strains can only be propagated and the infectivity content assayed by experimentally challenging conventional or transgenic animals. Robust cell culture systems are not available for any of the natural and only for a few of the experimental prion strains. Moreover, the pathogenesis of different transmissible spongiform encephalopathies (TSE) can be analysed systematically by using experimentally infected animals. While, in the beginning, animals belonging to the natural host species were used, more and more rodent model species have been established, mostly due to practical reasons. Nowadays, most of these experiments are performed using highly susceptible transgenic mouse lines expressing cellular prion proteins, PrP, from a variety of species like cattle, sheep, goat, cervidae, elk, hamster, mouse, mink, pig, and man. In addition, transgenic mice carrying specific mutations or polymorphisms have helped to understand the molecular pathomechanisms of prion diseases. Transgenic mouse models have been utilised to investigate the physiological role of PrP(C), molecular aspects of species barrier effects, the cell specificity of the prion propagation, the role of the PrP glycosylation, the mechanisms of the prion spread, the neuropathological roles of PrP(C) and of its abnormal isoform PrP(D) (D for disease) as well as the function of PrP Doppel. Transgenic mouse models have also been used for mapping of PrP regions involved in or required for the PrP conversion and prion replication as well as for modelling of familial forms of human prion diseases.     

Molecular profiling and comparison of field transmissible spongiform encephalopathy cases diagnosed in Catalunya

Molecular profiling of the proteinase K resistant prion protein (PrP(res)) is a technique that has been applied to the characterisation of transmissible spongiform encephalopathy (TSE) strains. An interesting example of the application of this technique is the ability to differentiate, at the experimental level, between bovine spongiform encephalopathy (BSE) and scrapie infection in sheep, and to distinguish between classical and atypical BSE and scrapie cases. Twenty-six BSE cases and two scrapie cases from an active TSE surveillance program and diagnosed at the PRIOCAT, TSE Reference Laboratory (Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona, Catalunya, Spain) were examined by Western blotting. Molecular profiling was achieved by comparing the glycosylation profile, deglycosylated PrP molecular weight and 6H4/P4 monoclonal antibody binding ratio. The results obtained during the characterisation of these field cases indicated an absence of atypical BSE cases in Catalunya.     

Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.     

First and second cattle passage of transmissible mink encephalopathy by intracerebral inoculation

To compare clinicopathologic findings of transmissible mink encephalopathy (TME) with other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie and chronic wasting disease [CWD]), two groups of calves (n = 4 each) were intracerebrally inoculated with TME agents from two different sources (mink with TME and a steer with TME). Two uninoculated calves served as controls. Within 15.3 months postinoculation, all animals from both inoculated groups developed clinical signs of central nervous system (CNS) abnormality; their CNS tissues had microscopic spongiform encephalopathy (SE); and abnormal prion protein (PrP(res)) as detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify TME PrP(res) but also develop clinical CNS signs and extensive lesions of SE. The latter has not been shown with other TSE agents (scrapie and CWD) similarly inoculated into cattle. The findings also suggest that the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) would detect TME in cattle should it occur naturally. However, it would be a diagnostic challenge to differentiate TME in cattle from BSE by clinical signs, neuropathology, or the presence of PrP(res) by IHC and WB.     

Strategies for eliminating PrP(c) as substrate for prion conversion and for enhancing PrP(Sc) degradation

Prion diseases are fatal neurodegenerative infectious disorders for which no therapeutic or prophylactic regimens exist. Our work aims to eliminate PrP(c) as substrate for the conversion into PrP(Sc) and to increase the cellular clearance capacity of PrP(Sc). In order to achieve the first objective, we used chemical compounds which interfere with the subcellular trafficking of PrP(c), e.g. by intracellular re-routing. Recently, we found that PrP(c) requires cholesterol for cell surface localisation. Treatment with mevinolin significantly reduced the amount of cell surface PrP(c) and led to its accumulation in the Golgi compartment. These data show that cholesterol is essential for the cell surface localisation of PrP(c), which is in turn known to be necessary for the formation of PrP(Sc). Another anti-prion strategy uses RNA and peptide aptamers directed against PrP(c). We have selected peptide aptamers using a constrained peptide library presented on the active site loop of the Escherichia coli protein TrxA in a Y2H screen. Several peptides reproducibly binding to PrP(c) in several assays were identified. Preliminary data indicate that selected peptide aptamers are able to interfere with prion propagation in prion-infected cells. To obtain additive effects we have tried to clarify cellular mechanisms that enable cells to clear prion infectivity. This goal was achieved by selective interference in intracellular signalling pathways which apparently also increase the cellular autophagy machinery. Finally, we have tried to establish an active auto-vaccination approach directed against PrP, which gave some positive preliminary results in the mouse system. This might open the door to classical immunological interference techniques.