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。     

Characterization of prion protein-enriched domains, isolated from rat cerebellar granule cells in culture

The biological functions of prion protein (PrP^C) and its possible interaction with other specific molecular membrane partners remain largely unknown. The aim of this study is to gain information on the molecular environment of PrP^C by analyzing the lipid and protein composition of a PrP^C-enriched membrane subfraction, called prion domain, PrD . This domain was obtained by immunoprecipitation of detergent-resistant microdomains (DRM) of rat cerebellar granule cells under conditions designed to preserve lipid-mediated membrane organization. The electrophoretic pattern of PrD , after staining with Coomassie blue, showed the enrichment of some protein bands in comparison with DRM. μLiquid cromatography-electrospray ionization-mass spectrometry (μLC-ESI-MS)/MS analysis showed that Thy-1 and different types of myosin were strongly enriched in PrD and, in a lesser extent, also OBCAM, LSAMP and tubulin, present altogether in a single band. Experiments using the chemical cross-linker BS³ suggested the existence of an interaction between PrP^C and neural cell adhesion molecule (NCAM). Concerning lipids, the comparison between PrD and DRM showed a similar phospholipid/sphingolipid ratio, a phospholipid/cholesterol ratio doubled, and a strong decrease of plasmenilethanolamine (19.7 ± 3.5% vs. 38.3 ± 1.2%). In conclusion, the peculiar lipid composition and in particular the presence of proteins involved in synaptic plasticity, cell adhesion, cytoskeleton regulation and signalling, suggest an important physiological role in neurons of Prion Domain.     

The effect of membrane domains on the G protein–phospholipase Cβ signaling pathway

The plasma membrane serves as a barrier to limit the exit and entry of components into and out of the cell, offering protection from the external environment. Communication between the cell and the external environment is mediated by multiple signaling pathways. While the plasma membrane was historically viewed as a lipid bilayer with freely diffusing proteins, the last decade has shown that the lipids and proteins in the plasma membrane are organized in a non-random manner, and that this organization can direct and modify various signaling pathways in the cell. In this review, we qualitatively discuss the ways that membrane domains can affect cell signaling. We then focus on how membrane domains can affect a specific signaling pathway – the G protein–phospholipase Cβ pathway and show how membrane domains can play an active role in directing or redirecting G protein signals.     

BECN1/Beclin 1 is recruited into lipid rafts by prion to activate autophagy in response to amyloid β 42

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid β_1–42 (Aβ₄₂)-induced autophagy activation through its interaction with BECN1. Treatment with Aβ₄₂ enhanced autophagy flux in neuronal cells. Aβ₄₂-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aβ₄₂. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aβ₄₂, defining a novel role of PRNP in the regulation of autophagy.     

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。     

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.     

Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro

Prion diseases are fatal and incurable neurodegenerative diseases of humans and animals. Despite years of research, no therapeutic agents have been developed that can effectively manage or reverse disease progression. Recently it has been identified that recombinant prion proteins (rPrP) expressed in bacteria can act as inhibitors of prion replication within the in vitro prion replication system protein misfolding cyclic amplification (PMCA). Here, within PMCA reactions amplifying a range of ruminant prions including distinct Prnp genotypes/host species and distinct prion strains, recombinant ovine VRQ PrP displayed consistent inhibition of prion replication and produced IC50 values of 122 and 171 nM for ovine scrapie and bovine BSE replication, respectively. These findings illustrate the therapeutic potential of rPrPs with distinct TSE diseases.     

Mammalian prions

Upon prion infection, abnormal prion protein (PrP^Sc) self-perpetuate by conformational conversion of α-helix-rich PrP^C into β sheet enriched form, leading to formation and deposition of PrP^Sc aggregates in affected brains. However the process remains poorly understood at the molecular level and the regions of PrP critical for conversion are still debated. Minimal amino acid substitutions can impair prion replication at many places in PrP. Conversely, we recently showed that bona fide prions could be generated after introduction of eight and up to 16 additional amino acids in the H2-H3 inter-helix loop of PrP. Prion replication also accommodated the insertions of an octapeptide at different places in the last turns of H2. This reverse genetic approach reveals an unexpected tolerance of prions to substantial sequence changes in the protease-resistant part which is associated with infectivity. It also demonstrates that conversion does not require the presence of a specific sequence in the middle of the H2-H3 area. We discuss the implications of our findings according to different structural models proposed for PrP^Sc and questioned the postulated existence of an N- or C-terminal prion domain in the protease-resistant region.     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Use of thermolysin in the diagnosis of prion diseases

The molecular diagnosis of prion diseases almost always involves the use of a protease to distinguish PrP^C from PrP^Sc and invariably the protease of choice is proteinase K. Here, we have applied the protease thermolysin to the diagnosis of animal prion diseases. This thermostable protease cleaves at the hydrophobic residues Leu, Ile, Phe, Val, Ala and Met, residues that are absent from the protease accessible aminoterminal region of PrP^Sc. Therefore, although thermolysin readily digests PrP^c into small protein fragments, full-length PrP^Sc is resistant to such proteolysis. This contrasts with proteinase K digestion where an aminoterminally truncated PrP^Sc species is produced, PrP^27–30. Thermolysin was used in the diagnosis of ovine scrapie and bovine spongiform encephalopathy and produced comparable assay sensitivity to assays using proteinase K digestion. Furthermore, we demonstrated the concentration of thermolysin-resistant PrP^Sc using immobilized metal-affinity chromatography. The use of thermolysin to reveal a full-length PrP^Sc has application for the development of novel immunodiagnostics by exploiting the wide range of commercially available immunoreagents and metal affinity matrices that bind the amino-terminal region of PrP. In addition, thermolysin provides a complementary tool to proteinase K to allow the study of the contribution of the amino-terminal domain of PrP^Sc to disease pathogenesis.     

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.     

Aggregation of cellular prion protein is initiated by proximity-induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.     

朊蛋白的细胞生物学研究

朊蛋白病是人和牛羊等哺乳动物所患的致命性的神经系统变性疾病,它是由机体内正常的朊蛋白改变构象后所引起的疾病。本综述对朊蛋白在细胞生物学领域的认知和理解进行了归纳总结,阐述了正常和异常朊蛋白的翻译、表达、定位、裂解、转化等一系列过程,是对疾病本质的有益探索。     

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.