朊蛋白相关蛋白Shadoo与朊病毒病

朊病毒病,即传染性海绵状脑病（transmissible spongiform encephalopathies, TSEs）,是一类传染性、致死性神经退行性疾病。在朊病毒病的病理过程中,细胞正常朊蛋白PrP。转化为异常构象的PrP是至关重要的,但是PrP‘的正常生理功能仍不清楚。国外学者利用比较基因组学发现了-个新的朊蛋白相关蛋白-shadoo（Sho）。Sho与PrP。在氨基酸序列和细胞定位的相似性及主要在脑组织表达,使它成为-个非常值得研究的PrP相关蛋白。对Sho可能存在的与PrP。重叠的功能甚至直接相互作用的研究工作,将对今后揭示PrPc正常生理功能以及揭示Pfion病发病机制具有重要现实意义。     

Functions of prion protein PrPc

It is now well established that both normal and pathological (or scrapie) isoforms of prion protein, PrPc and PrPsc respectively, are involved in the development and progression of various forms of neurodegenerative diseases, including scrapie in sheep, bovine spongiform encephalopathy (or "mad cow disease") and Creutzfeldt-Jakob disease in human, collectively known as prion diseases. The protein PrPc is highly expressed in the central nervous system in neurons and glial cells, and also present in non-brain cells, such as immune cells or epithelial and endothelial cells. Identification of the physiological functions of PrPc in these different cell types thus appears crucial for understanding the progression of prion diseases. Recent studies highlighted several major roles for PrPc that may be considered in two major domains : (1) cell survival (protection against oxidative stress and apoptosis) and (2) cell adhesion. In association with cell adhesion, distinct functions of PrPc were observed, depending on cell types : neuronal differentiation, epithelial and endothelial barrier integrity, transendothelial migration of monocytes, T cell activation. These observations suggest that PrPc functions may be particularly relevant to cellular stress, as well as inflammatory or infectious situations.     

Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

Background  

A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions.     

利用PMCA技术实验不同剂量MCT联合DTT对PrP~(sc)转化的抑制效应

蛋白质错误折叠循环扩增(protein misfolding cyclic amplification,PMCA)技术,是一种具有感染性的朊蛋白(PrPsc)体外扩增的技术,可用于抑制细胞型朊蛋白(PrPc)向PrPsc转化药物的筛选.本研究在Saborio等提供的方法基础上,成功优化了最佳扩增时间,利用优化的PMCA技术实验了不同剂量氮芥(mechlorethamine,MCT)联合二硫苏糖醇(dithiothreitol,DTT)对PrPsc转化的抑制效应.结果发现,MCT联合DTT能体外抑制PrPc向PrPsc的转化,抑制作用具有明显的量效关系.利用针对人PrPc不同结构域的抗体发现,MCT联用DTT可使抗体6H4的抗原表位隐蔽,该表位位于PrPc第1个α螺旋区内,是构象转化的主要部位.对其机制的深入探讨,将有助于新一类可传播性海绵状脑病(transmissible spongiform encephalopathy,TSE)治疗药物的研发.     

Cellular prion protein is expressed in a subset of neuroendocrine cells of the rat gastrointestinal tract.

Prion diseases are believed to develop from the conformational change of normal cellular prion protein (PrPc) to a pathogenic isoform (PrPsc). PrPc is present in both the central nervous system and many peripheral tissues, although protein concentration is significantly lower in non-neuronal tissues. PrPc expression is essential for internalization and replication of the infectious agent. Several works have pointed to the gastrointestinal (GI) tract as the principal site of entry of PrPsc, but how passage through the GI mucosa occurs is not yet known. Here we studied PrPc expression using Western blot, RT-PCR, and immunohistochemistry in rat GI tract. PrPc mRNA and protein were detected in corpus, antrum, duodenum, and colon. Immunoreactivity was found in scattered cells of the GI epithelium. With double immunofluorescence, these cells have been identified as neuroendocrine cells. PrPc immunostaining was found in subsets of histamine, somatostatin (Som), ghrelin, gastrin (G), and serotonin (5HT) cells in stomach. In small and large bowel, PrPc cells co-localized with subpopulations of 5HT-, Som-, G-, and peptide YY-immunolabeled cells. Our results provide evidence for a possible and important role of endocrine cells in the internalization of PrPsc from gut lumen.     

Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.     

The cellular prion protein: a new partner of the lectin CBP70 in the nucleus of NB4 human promyelocytic leukemia cells.

Prion diseases are characterized by the presence of an abnormal isoform of the cellular prion protein (PrPc) whose physiological role still remains elusive. To better understand the function of PrPc, it is important to identify the different subcellular localization(s) of the protein and the different partners with which it might be associated. In this context, the PrPc-lectins interactions are investigated because PrPc is a sialoglycoprotein which can react with lectins which are carbohydrate-binding proteins. We have previously characterized a nuclear lectin CBP70 able to recognize N-acetyl-beta-D-glucosamine residues in HL60 cells. Using confocal immunofluorescence, flow-cytofluorometry, and Western-blotting, we have found that PrPc is expressed in the nucleus of the NB4 human promyelocytic leukemia cell line. It was also found that the lectin CBP70 is localized in NB4 cell nuclei. Moreover, several approaches revealed that PrPc and CBP70 are colocalized in the nucleus. Immunoprecipitation experiments showed that these proteins are coprecipitated and interact via a sugar-dependent binding moiety. In conclusion, PrPc and CBP70 are colocalized in the nuclear compartment of NB4 cells and this interaction may be important to better understand the biological function and possibly the conversion process of PrPc into its pathological form (PrPsc).     

Prion metal interaction: Is prion pathogenesis a cause or a consequence of metal imbalance?

Functional role of cellular prion protein (PrPc) has been hypothesized to be in metal homeostasis and providing cells with a superoxide dismutase (SOD)-like activity to escape damage by reactive oxygen species (ROS). PrPc interacts with a range of divalent metal ions and undergoes Cu²⁺ as well as Zn²⁺-associated endocytosis, thereby maintaining homeostasis of these and other metal ions. Conformational change to a β-sheet rich, protease resistant entity, reminiscent of the disease-associated scrapie form called PrPsc, has been found to be induced by interaction of PrPc with metal ions like Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺. This review compiles data from various experimental studies of the interaction of metals with PrPc. The effect of metal ions on the expression and conformation of the prion protein is described in detail with emphasis on their possible physiological and pathogenic role. Further, a hypothesis is presented where attainment of altered conformation by metal-bound PrPc has been viewed as a deleterious consequence of efforts made by cells to maintain metal homeostasis. Thus, PrPc presumably sacrifices itself by converting into PrPsc form in an attempt to protect cells from the toxicity of metal imbalance. Finally, possible reasons for contradictions reported in the literature on the subject are explored and experimental approaches to resolve the same are suggested.     

Human and animal spongiform encephalopathies are the result of chronic autoimmune attack in the CNS: a novel medical theory supported by overwhelming experimental evidence

Spongiform encephalopathies, also called "prion diseases", are fatal degenerative diseases of the central nervous system which can occur in animals (such as the "mad cow disease" in cattle) and also in humans. This paper presents a novel medical theory concerning the pathogenic mechanisms for various human and animal spongiform encephalopathies. It is hypothesized that various forms of prion diseases are essentially autoimmune diseases, resulting from chronic autoimmune attack of the central nervous system. A key step in the pathogenic process leading towards the development of spongiform encephalopathies involves the production of specific autoimmune antibodies against the disease-causing prion protein (PrPsc) and possibly other immunogenic macromolecules present in the brain. As precisely explained in this paper, the autoimmune antibodies produced against PrPsc are responsible for the conversion of the normal cellular prion protein (PrPc) to PrPsc, for the accumulation of PrPsc in the brain and other peripheral tissues, and also for the initiation of an antibody-mediated chronic autoimmune attack of the central nervous system neurons, which would contribute to the development of characteristic pathological changes and clinical symptoms associated with spongiform encephalopathies. The validity and correctness of the proposed theory is supported by an overwhelming body of experimental observations that are scattered in the biomedical literature. In addition, the theory also offers practical new strategies for early diagnosis, treatment, and prevention of various human and animal prion diseases.     

Cellular cholesterol enrichment prevents prion peptide-induced neuron cell damages

The prion diseases are neurodegenerative disorders characterized by the conversion of the PrPc (normal cellular prion) to the PrPsc (misfolded isoform). The accumulation of PrPsc within the central nervous system (CNS) leads to neurocytotoxicity by increasing oxidative stress. In addition, many neurodegenerative disorders including prion, Parkinson’s and Alzheimer’s diseases may be regulated by cholesterol homeostasis. The effects of cholesterol balance on prion protein-mediated neurotoxicity and ROS (reactive oxygen species) generation were the focus of this study. Cholesterol treatment inhibited PrP (106-126)-induced neuronal cell death and ROS generation in SH-SY5Y neuroblastoma cells. In addition, the PrP (106-126)-mediated increase of p53, p-p38, p-ERK and the decrease of Bcl-2 were blocked by cholesterol treatment. These results indicated that cellular cholesterol enrichment is a key regulator of PrP-106-126-mediated oxidative stress and neurotoxicity. Taken together, the results of this study suggest that modulation of cellular cholesterol appears to prevent the neuronal cell death caused by prion peptides.     

cDNA cloning of a novel heterogeneous nuclear ribonucleoprotein gene homologue in Caenorhabditis elegans using hamster prion protein cDNA as a hybridization probe.

The mammalian prion protein (PrPc) is a cellular protein of unknown function, an altered isoform of which (PrPsc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. The evolutionary conservation of the PrP gene has been reported in the genomes of many vertebrates as well as certain invertebrates. In the genome of nematode Caenorhabditis elegans, the sequence capable of hybridizing with the mammalian PrP cDNA probe has been demonstrated, predicting the presence of the PrP gene homologue in C.elegans. In this study, Southern analysis with the hamster PrP cDNA (HaPrP) probe confirmed the previous observation. Moreover, Northern analysis revealed that the sequence is actively transcribed in adult worms. Thus, we screened C.elegans cDNA libraries with the HaPrP probe and isolated a cDNA that hybridizes to the same sequence in C.elegans that hybridized with the HaPrP probe in the Southern and Northern analyses. The deduced amino acid sequence of this cDNA, however, is substantially homologous with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins rather than mammalian PrPc. The hnRNPs contain the glycine-rich domain in the C-terminal half of the molecule, which also seemed to be in PrPc at the N-terminal half of the molecule. Both of the glycine-rich domains are composed of tracts with high G + C content, indicating that these tracts may due to the hybridizing signals. These results suggest that this cDNA clone is derived from a novel hnRNP gene homologue in C.elegans but not from a predicted PrP gene homologue.     

High frequency occurrence of 1-OPRD variant of PRNP gene in gastric cancer cell lines and Chinese population with gastric cancer

The prion protein gene PRNP encodes PrPc and PrPsc, causing a number of neurological disorders. Approximately 10-15% of human prion disease is inherited and more than 20 pathogenic mutations have been found. Most of the genetic alterations are point mutations, with the exception of genetic insertions of one to nine extra octapeptide repeats occurring in the important octapeptide-coding region. Our previous work showed that PrPc was overexpressed in gastric cancer. We wondered whether mutations of PrPc existed in human gastric cancer. DNA sequencing and gel electrophoresis were used to determine the possible mutation of PrPc in patients and cell lines of gastric cancer. We found that 1-OPRD (one octapeptide-repeat deletion) homozygosity or heterozygosity exists in several gastric cancer cell lines, e.g. MKN28 and KatoIII are homozygous for 1-OPRD, and SGC7901 and BGC-823 are heterozygous for 1-OPRD. The mutation frequency in tissues of gastric cancer cases is significantly higher than that in the common population (p<0.05). All positive cases in gastric cancer were found to be heterozygous for 1-OPRD. Further study of the variant may be helpful in understanding the mechanisms of occurrence and development of clinical gastric carcinoma as well as the biology of the mysterious gene PRNP.     

New insights into early sequential PrPsc accumulation in scrapie infected mouse brain evidenced by the use of streptomycin sulfate

To investigate the amplifying potentialities of streptomycin sulfate in the immunohistochemical (IHC) detection of the abnormal prion protein (PrPsc), we used a sequential brain sampling from C506M3 scrapie strain inoculated C57Bl/6 mice. The weekly removed brains, from 7 to 63 days post intra-cranial inoculation were analysed using PrPsc IHC. The introduction of streptomycin sulfate, a technique developed for accurate cellular and regional mapping of PrPsc deposition in several animal TSEs, revealed a substantial amplifying effect and a clear specific PrPsc detection as early as 28 days post inoculation. The location of the first detected PrPsc deposits suggests a possible involvement of the cerebrospinal fluid in the early dissemination of the infectious agent. The meaning of these newly accessible PrPsc deposits is discussed in relation to a possible nascent form of PrPsc molecules detected in situ for the first time. Altogether, these findings argue that this method can be highly useful to study the early stages after infection with prion agents.     

Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches

Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n=1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.     

Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein

As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79-92 (SAF 34 and SAF 32 Mo-Abs); the 6H4 antibody reacts with a specific peptide comprising the 144-152 amino acids, and the 12F10 MoAb recognises the sequence 142-160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79-92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy.     

Efficient and specific down-regulation of prion protein expression by RNAi

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.     

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.     

Distribution and submicroscopic immunogold localization of cellular prion protein (PrPc) in extracerebral tissues

In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.     

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.     

Axonal transport of the cellular prion protein is increased during axon regeneration

The cellular prion protein, PrPc, is a glycosylphosphatidylinositol-anchored cell surface glycoprotein and a protease-resistant conformer of the protein may be the infectious agent in transmissible spongiform encephalopathies. PrPc is localized on growing axons in vitro and along fibre bundles that contain elongating axons in developing and adult brain. To determine whether the growth state of axons influenced the expression and axonal transport of PrPc, we examined changes in the protein following post-traumatic regeneration in the hamster sciatic nerve. Our results show (1) that PrPc in nerve is significantly increased during nerve regeneration; (2) that this increase involves an increase in axonally transported PrPc; and (3) that the PrPc preferentially targeted for the newly formed portions of the regenerating axons consists of higher molecular weight glycoforms. These results raise the possibility that PrPc may play a role in the growth of axons in vivo, perhaps as an adhesion molecule interacting with the extracellular environment through specialized glycosylation.