Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone.

A conformational transition between the normal cellular prion protein (PrPC) and the beta-sheet-rich pathological isoform (PrPSc) is a central event in the pathogenesis of spongiform encephalopathies. The prion infectious agent seems to contain mainly, if not exclusively, PrPSc, which has the ability to propagate its abnormal conformation by transforming the host PrPC into the pathological isoform. We have developed an in vitro system to induce the PrPC --> PrPSc conversion by incubating a cell-lysate containing mouse PrPC with partially purified mouse PrPSc. After 48 h of incubation with a 10-fold molar excess of PrPSc, the cellular protein acquired PK-resistance resembling a PrPSc-like state. Time course experiments suggest that the conversion follows a stepwise mechanism involving kinetic intermediates. The conversion was induced by PrPSc extracted from mice infected with two different prion strains, each propagating its characteristic Western blot profile. The latter results and the fact that all the cellular components are present in the conversion reaction suggest that PrPC-PrPSc interaction is highly specific and required for the conversion. No transformation was observed under the same conditions using purified proteins without cell-lysate. However, when PrPC-depleted cell-lysate was added to the purified proteins the conversion was recovered. These findings provide direct evidence for the participation of a chaperone-like activity involved in catalyzing the conversion of PrPC into PrPSc.     

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.     

Hsp70 binds to PrPC in the process of PrPC release via exosomes from THP-1 monocytes

PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the surface of a number of peripheral blood cells. PrPC must be present for the generation and propagation of pathogenic conformer [PrPSc (scrapie prion protein)], which is a conformational conversion form of PrPC and has a central role in transmissible spongiform encephalopathies. It is important to determine the transportation mechanism of normal PrPC between cells. Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. We have identified that THP-1 monocytes can secrete exosomes to culture medium, and the secreted exosomes can bear PrPC. We also found that Hsp70 interacts with PrPC not only in intracellular environment, but in the secreted exosomes. However, the specific markers of exosomes, Tsg101 and flotillin-1, were found with no interaction with PrPC. Our results demonstrated that PrPC can be released from THP-1 monocytes via secreted exosomes, and in this process, Hsp70 binds to PrPC, which suggests that Hsp70 may play a potential functional role in the release of PrPC.     

朊粒蛋白PrP~(Sc)寡聚体的形成与跨膜毒性

朊粒蛋白(prionprotein,PrP)传染致病机制一直是朊粒(prion)研究领域的焦点.由正常型朊粒蛋白(PrPC)向致病型朊粒蛋白(PrPSc)的转变是致病的关键步骤.本文综述了近年来PrPC向PrPSc转变的结构变化特征、PrPSc由单体形成寡聚体的组装机制、以及PrPSc寡聚体的跨膜机制与细胞毒性间的关系等方面的研究进展.     

朊病毒和朊病毒病研究进展

朊病毒病即海绵状脑病,是人和动物中的一类致死性中央神经系统疾病,近几年来在朊病毒病的致病机制及其诊断技术和防治策略方面取得了很大的研究进展.     

Prion and TNFα: TAC(E)it agreement between the prion protein and cell signaling

Prion diseases are rare and fatal neurodegenerative disorders that occur when the cellular prion protein (PrPC) is converted into a conformationally modified isoform that originates the novel infectious agent, called prion. Although much information is now available on the different routes of prion infection, both the mechanisms underlying prion neurotoxicity and the physiologic role of PrPC remain unclear. By use of a novel paradigm, we have shown in a recent paper that - following a myotoxin-induced degenerative challenge - PrPC is implicated in the morphogenesis of the skeletal muscle of adult mice. PrPC accomplished this task by modulating signaling pathways central to the myogenic process, in particular the p38 kinase pathway. The possibility that PrPC acts in cell signaling has already been suggested after in vitro studies. Using our in vivo approach, we have instead provided proof of the physiologic relevance of PrPC commitment in signaling events, and that PrPC likely performed the task by controlling the activity of the enzyme (TACE) secreting the signaling TNF-α molecule. After a brief summary of our data, here we will discuss the suggestion, arising from our and other recent findings, implying that regulation of TACE, and of other members of the protease family TACE belongs to, may be exploited by PrPC in different cell contexts. Notably, this advancement of knowledge on PrPC physiology could also shed light on the defense mechanisms against the onset of a more common neurodegenerative disorder than prion disease, such as Alzheimer disease.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

PrP的胞内运输与朊病毒病

朊病毒病,即传染性海绵状脑病（transmissible spongiform encephalopathies,TSEs）,是一类致死性的神经退行性疾病,存在散发性、感染性和遗传性3种形式。在朊病毒病的病理过程中,细胞正常朊蛋白PrPc（cellular PrP）转化为异常构象的PrP^Sc（scrapie PrP）是至关重要的,但是朊病毒的增殖如何导致神经元凋亡仍不清楚。PrPc的胞内运输在朊病毒病中发挥重要作用,朊病毒感染后PrP^C转化为PrP^Sc,及遗传性朊病毒病中PrP突变可能影响PrP的生物合成、亚细胞定位及转运过程,通过干扰PrP^C的正常功能或产生毒性中间体而导致神经系统病变。现对近年来关于PrP胞内运输在朊病毒病中的作用进行综述。     

A naturally occurring C-terminal fragment of the prion protein (PrP) delays disease and acts as a dominant-negative inhibitor of PrPSc formation

The cellular prion protein (PrPC) undergoes constitutive proteolytic cleavage between residues 111/112 to yield a soluble N-terminal fragment (N1) and a membrane-anchored C-terminal fragment (C1). The C1 fragment represents the major proteolytic fragment of PrPC in brain and several cell types. To explore the role of C1 in prion disease, we generated Tg(C1) transgenic mice expressing this fragment (PrP(Δ23-111)) in the presence and absence of endogenous PrP. In contrast to several other N-terminally deleted forms of PrP, the C1 fragment does not cause a spontaneous neurological disease in the absence of endogenous PrP. Tg(C1) mice inoculated with scrapie prions remain healthy and do not accumulate protease-resistant PrP, demonstrating that C1 is not a substrate for conversion to PrPSc (the disease-associated isoform). Interestingly, Tg(C1) mice co-expressing C1 along with wild-type PrP (either endogenous or encoded by a second transgene) become ill after scrapie inoculation, but with a dramatically delayed time course compared with mice lacking C1. In addition, accumulation of PrPSc was markedly slowed in these animals. Similar effects were produced by a shorter C-terminal fragment of PrP(Δ23-134). These results demonstrate that C1 acts as dominant-negative inhibitor of PrPSc formation and accumulation of neurotoxic forms of PrP. Thus, C1, a naturally occurring fragment of PrPC, might play a modulatory role during the course of prion diseases. In addition, enhancing production of C1, or exogenously administering this fragment, represents a potential therapeutic strategy for the treatment of prion diseases.     

传染性海绵状脑病的诊断进展和存在的问题

传染性海绵状脑病是由朊病毒引起的人和多种哺乳动物以神经退行性变化为主要特征的一种慢性致死性传染病。引起这类疾病的病原因子是一种编码宿主蛋白的PrPC转变为异常的PrPSC沉积在大脑,导致传染性海绵状脑病的发生。本文从临床症状识别、组织病理学诊断、致病性朊蛋白检测、生物学测定以及毒株鉴定等几个方面作一回顾和总结,为揭示朊病毒疾病致病机理和诊断研究提供借鉴。     

Integrity of H1 helix in prion protein revealed by molecular dynamic simulations to be especially vulnerable to changes in the relative orientation of H1 and its S1 flank

In the template-assistance model, normal prion protein (PrPC), the pathogenic cause of prion diseases such as Creutzfeldt-Jakob in human, bovine spongiform encephalopathy in cow, and scrapie in sheep, converts to infectious prion (PrPSc) through an autocatalytic process triggered by a transient interaction between PrPC and PrPSc. Conventional studies suggest the S1-H1-S2 region in PrPC to be the template of S1-S2 β-sheet in PrPSc, and the conformational conversion of PrPC into PrPSc may involve an unfolding of H1 in PrPC and its refolding into the β-sheet in PrPSc. Here we conduct a series of simulation experiments to test the idea of transient interaction of the template-assistance model. We find that the integrity of H1 in PrPC is vulnerable to a transient interaction that alters the native dihedral angles at residue Asn¹⁴³, which connects the S1 flank to H1, but not to interactions that alter the internal structure of the S1 flank, nor to those that alter the relative orientation between H1 and the S2 flank.     

Atypical effect of salts on the thermodynamic stability of human prion protein

Prion diseases are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Previous studies indicate that salts promote conformational conversion of the recombinant prion protein into a PrPSc-like form. To gain insight into the mechanism of this effect, here we have studied the influence of a number of salts (sodium sulfate, sodium fluoride, sodium acetate, and sodium chloride) on the thermodynamic stability of the recombinant human prion protein. Chemical unfolding studies in urea show that at low concentrations (below approximately 50 mm), all salts tested significantly reduced the thermodynamic stability of the protein. This highly unusual response to salts was observed for both the full-length prion protein as well as the N-truncated fragments huPrP90-231 and huPrP122-231. At higher salt concentrations, the destabilizing effect was gradually reversed, and salts behaved according to their ranking in the Hofmeister series. The present data indicate that electrostatic interactions play an unusually important role in the stability of the prion protein. The abnormal effect of salts is likely because of the ion-induced destabilization of salt bridges (Asp144-Arg148 and/or Asp147-Arg151) in the extremely hydrophilic helix 1. Contrary to previous suggestions, this effect is not due to the interaction of ions with the glycine-rich flexible N-terminal region of the prion protein. The results of this study suggest that ionic species present in the cellular environment may control the PrPC to PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.     

In vitro generation of infectious scrapie prions

Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the cellular prion protein (PrPC). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.     

Functional implication of cellular prion protein in antigen-driven interactions between T cells and dendritic cells

The cellular prion protein (PrPC) is a host-encoded, GPI-anchored cell surface protein, expressed on a wide range of tissues including neuronal and lymphoreticular cells. PrPC may undergo posttranslational conversion, giving rise to scrapie PrP, the pathogenic conformer considered as responsible for prion diseases. Despite intensive studies, the normal function of PrPC is still enigmatic. Starting from microscope observations showing an accumulation of PrPC at the sites of contact between T cells and Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC interactions. Whereas the absence of PrPC on the DC results in a reduced allogeneic T cell response, its absence on the T cell partner has no apparent effect upon this response. Therefore, PrPC seems to fulfill different functions on the two cell partners forming the synapse. In contrast, PrPC mobilization by Ab reduces the stimulatory properties of DC and the proliferative potential of responding T cells. The contrasted consequences, regarding T cell function, between PrPC deletion and PrPC coating by Abs, suggests that the prion protein acts as a signaling molecule on T cells. Furthermore, our results show that the absence of PrPC has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neurological considerations, some of the evolutionary constraints that may have contributed to the conservation of the Prnp gene in mammalians, could be of immunological origin.     

Protease-resistant and detergent-insoluble prion protein is not necessarily associated with prion infectivity.

PrPSc, an abnormal isoform of PrPC, is the only known component of the prion, an agent causing fatal neurodegenerative disorders such as bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). It has been postulated that prion diseases propagate by the conversion of detergent-soluble and protease-sensitive PrPC molecules into protease-resistant and insoluble PrPSc molecules by a mechanism in which PrPSc serves as a template. We show here that the chemical chaperone dimethyl sulfoxide (Me2SO) can partially inhibit the aggregation of either PrPSc or that of its protease-resistant core PrP27-30. Following Me2SO removal by methanol precipitation, solubilized PrP27-30 molecules aggregated into small and amorphous structures that did not resemble the rod configuration observed when scrapie brain membranes were extracted with Sarkosyl and digested with proteinase K. Interestingly, aggregates derived from Me2SO-solubilized PrP27-30 presented less than 1% of the prion infectivity obtained when the same amount of PrP27-30 in rods was inoculated into hamsters. These results suggest that the conversion of PrPC into protease-resistant and detergent-insoluble PrP molecules is not the only crucial step in prion replication. Whether an additional requirement is the aggregation of newly formed proteinase K-resistant PrP molecules into uniquely structured aggregates remains to be established.     

Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity

It has been estimated that cerebrospinal fluid (CSF) contains approximately 80 proteins that significantly increase or decrease in response to various clinical conditions. Here we have evaluated the CSF protein PrPC (cellular prion protein) for possible increases or decreases following spinal cord injury. The physiological function of PrPC is not yet completely understood; however, recent findings suggest that PrPC may have neuroprotective properties. Our results show that CSF PrPC is decreased in spinal cord injured patients 12 hours following injury and is absent at 7 days. Given that normal PrPC has been proposed to be neuroprotective we speculate that the decrease in CSF PrPC levels may influence neuronal cell survival following spinal cord injury.     

The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.     

Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein

Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121-231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121-231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121-231) and similar to full-length PrP(23-231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation. The thermodynamic stabilities of the variants do not correlate with specific disease phenotypes. Five of the amino acid replacements destabilize PrP(121-231), but the other variants have the same stability as the wild-type protein. These data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.     

The role of electrostatic interaction in triggering the unraveling of stable helix 1 in normal prion protein. A molecular dynamics simulation investigation

The conversion of normal prion protein (PrPC) into scrapie isoform (PrPSc) is a key event in the pathogenesis of prion diseases. However, the conversion mechanism has given rise to much controversy. For instance, there is much debate on the behavior of helix 1 (H1) in the conversion. A series of experiments demonstrated that H1 in isolated state was very stable under a variety of conditions. But, other experiments indicated that helices 2 and 3 rather than H1 were retained in PrPSc. In this paper, molecular dynamics (MD) simulation is employed to investigate the dynamic behavior of H1. It is revealed that although the helix 1 of Human PrPC (HuPrPC) is very stable in the isolated state, it becomes unstable when incorporated into native HuPrPC, which likely results from the long-range electrostatic interaction between Asp147 and Arg208 located in the helices 1 and 3, respectively. This explanation is supported by experimental evaluation and MD simulation on D147N mutant of HuPrPC that the mutant becomes a little more stable than the wild type HuPrPC. This finding not only help to reconcile the existing debate on the role of helix 1 in the PrPC-->PrPSc transition, but also reveals a possible mechanism for triggering the PrPC-->PrPSc conversion.     

KDEL-tagged anti-prion intrabodies impair PrP lysosomal degradation and inhibit scrapie infectivity

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into the infectious scrapie isoform (PrPSc). We have recently demonstrated that anti-prion intrabodies targeted to the lumen of the endoplasmic reticulum provide a simple and effective means to inhibit the transport of PrPC to the cell surface. Here, we report that they completely block the traffic of mature full-length PrPC molecules, impair prion lysosomal degradation, and interfere with the early phase of scrapie formation. Since anti-prion intrabodies efficiently block PrPSc accumulation in vitro, we investigated whether they could also antagonize scrapie infectivity in vivo. We found that mice intracerebrally injected with KDEL-8H4-NGF-differentiated PC12 cells infected with scrapie neither develop scrapie clinical signs nor brain damage. Furthermore, no protease-resistant PrPSc is detectable in brains of inoculated animals. These results indicate that anti-prion intrabody strategy may be effective against prion infection.