Probing structural differences between PrP(C) and PrP(Sc) by surface nitration and acetylation: evidence of conformational change in the C-terminus

We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride (Ac(2)O), which specifically target accessible tyrosine and lysine residues, respectively, to modify recombinant Syrian hamster PrP(90-231) [rSHaPrP(90-231)] and SHaPrP 27-30, the proteinase K-resistant core of PrP(Sc) isolated from brain of scrapie-infected Syrian hamsters. Our aim was to find locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin and subsequent analysis by mass spectrometry (MALDI-TOF). Several differences in chemical reactivity were observed. With TNM, the most conspicuous reactivity difference seen involves peptide E(221)-R(229) (containing Y(225) and Y(226)), which in rSHaPrP(90-231) was much more extensively modified than in SHaPrP 27-30; peptide H(111)-R(136), containing Y(128), was also more modified in rSHaPrP(90-231). Conversely, peptides Y(149)-R(151), Y(157)-R(164), and R(151)-Y(162) suffered more extensive modification in SHaPrP 27-30. Acetic anhydride modified very extensively peptide G(90)-K(106), containing K(101), K(104), K(106), and the amino terminus, in both rSHaPrP(90-231) and SHaPrP 27-30. These results suggest that (1) SHaPrP 27-30 exhibits important conformational differences in the C-terminal region with respect to rSHaPrP(90-231), resulting in the loss of solvent accessibility of Y(225) and Y(226), very solvent-exposed in the latter conformation; because other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in SHaPrP 27-30. (2) On the other hand, tyrosines contained in the stretch spanning approximately Y(149)-R(164) are more accessible in SHaPrP 27-30, suggesting rearrangements in α-helix H1 and the short β-sheet of rSHaPrP(90-231). (3) The amino-terminal region of SHaPrP 27-30 is very accessible. These data should help in the validation and construction of structural models of PrP(Sc).     

Oxidation of methionine residues in the prion protein by hydrogen peroxide

Reaction of H(2)O(2) with the recombinant SHa(29-231) prion protein resulted in rapid oxidation of multiple methionine residues. Susceptibility to oxidation of individual residues, assessed by mass spectrometry after digestion with CNBr and lysC, was in general a function of solvent exposure. Met 109 and Met 112, situated in the highly flexible amino terminus, and key residues of the toxic peptide PrP (106-126), showed the greatest susceptibility. Met 129, a residue located in a polymorphic position in human PrP and modulating risk of prion disease, was also easily oxidized, as was Met 134. The structural effect of H(2)O(2)-induced methionine oxidation on PrP was studied by CD spectroscopy. As opposed to copper catalyzed oxidation, which results in extensive aggregation of PrP, this reaction led only to a modest increase in beta-sheet structure. The high number of solvent exposed methionine residues in PrP suggests their possible role as protective endogenous antioxidants.     

Assembly of natural and recombinant prion protein into fibrils

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.     

Toxicity of novel C-terminal prion protein fragments and peptides harbouring disease-related C-terminal mutations.

Mice expressing a C-terminal fragment of the prion protein instead of wild-type prion protein die from massive neuronal degeneration within weeks of birth. The C-terminal region of PrPc (PrP121-231) expressed in these mice has an intrinsic neurotoxicity to cultured neurones. Unlike PrPSc, which is not neurotoxic to neurones lacking PrPc expression, PrP121-231 was more neurotoxic to PrPc-deficient cells. Human mutations E200K and F198S were found to enhance toxicity of PrP121-231 to PrP-knockout neurones and E200K enhanced toxicity to wild-type neurones. The normal metabolic cleavage point of PrPc is approximately amino-acid residue 113. A fragment of PrPc corresponding to the whole C-terminus of PrPc (PrP113-231), which is eight amino acids longer than PrP121-231, lacked any toxicity. This suggests the first eight amino residues of PrP113-121 suppress toxicity of the toxic domain in PrP121-231. Addition to cultures of a peptide (PrP112-125) corresponding to this region, in parallel with PrP121-231, suppressed the toxicity of PrP121-231. These results suggest that the prion protein contains two domains that are toxic on their own but which neutralize each other's toxicity in the intact protein. Point mutations in the inherited forms of disease might have their effects by diminishing this inhibition.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.     

Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.     

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

Cryptic epitopes in N-terminally truncated prion protein are exposed in the full-length molecule: dependence of conformation on pH

Prion diseases are diseases of protein conformation. Structure-dependent antibodies have been sought to probe conformations of the prion protein (PrP) resulting from environmental changes, such as differences in pH. Despite the absence of such antibodies for full-length PrP, a recombinant Fab (D13) and a Fab derived from mAb 3F4 showed pH-dependent reactivity toward epitopes within the N-terminus of N-terminally truncated PrP(90-231). Refolding and maintaining this protein at pH > or =5.2 before immobilization on an ELISA plate inhibited reactivity relative to protein exposed to pH < or =4.7. The reactivity was not affected by pH changes after immobilization, showing retention of conformation after binding to the plate surface, although guanidine hydrochloride at 1.5-2 M was able to expose the cryptic epitopes after immobilization at pH > or =5.2. The alpha-helical CD spectrum of PrP(90-231) refolded at pH 5.5 was reduced somewhat by these pH changes, with a minor shift toward beta-sheet at pH 4 and then toward coil at pH 2. No covalent changes were caused by the pH differences. This pH dependence suggests titration of an acidic region that might inhibit the N-terminal epitopes. A similar pH dependence for a monoclonal antibody reactive to the central region identified an acidic region incorporating Glu152 as a significant participant.     

Recombinant human prion protein fragment 90-231, a useful model to study prion neurotoxicity

Transmissible spongiform encephalopathies (TSE), or prion diseases, are a group of fatal neurodegenerative disorders of animals and humans. Human diseases include Creutzfeldt-Jakob (CJD) and Gerstmann-Straussler-Scheinker (GSSD) diseases, fatal familial insomnia, and Kuru. Human and animal TSEs share a common histopathology with a pathognomonic triad: spongiform vacuolation of the grey matter, neuronal death, glial proliferation, and, more inconstantly, amyloid deposition. According to the "protein only" hypothesis, TSEs are caused by a unique post-translational conversion of normal, host-encoded, protease-sensitive prion protein (PrP(sen) or PrP(C)) to an abnormal disease-associated isoform (PrP(res) or PrP(Sc)). To investigate the molecular mechanism of neurotoxicity induced by PrP(Sc) we developed a protocol to obtain millimolar amounts of soluble recombinant polypeptide encompassing the amino acid sequence 90-231 of human PrP (hPrP90-231). This protein corresponds to the protease-resistant prion protein fragment that originates after amino-terminal truncation. Importantly, hPrP90-231 has a flexible backbone that, similar to PrP(C), can undergo to structural rearrangement. This peptide, structurally resembling PrP(C), can be converted in a PrP(Sc)-like conformation, and thus represents a valuable model to study prion neurotoxicity. In this article we summarized our experimental evidence on the molecular and structural mechanisms responsible of hPrP90-231 neurotoxicity on neuroectodermal cell line SHSY5Y and the effects of some PrP pathogen mutations identified in familial TSE.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

Detection of prion epitopes on PrP and PrP of transmissible spongiform encephalopathies using specific monoclonal antibodies to PrP

Amino acid residues 90-120 of the prion protein (PrP) are likely to be critical for the conversion of PrP(c) to PrP(sc) in the transmissible spongiform encephalopathies. We raised 10 monoclonal antibodies against the 90-120 amino acid region, mapped the epitope specificity of these anti-PrP antibodies, and investigated the expression of epitopes recognized by the antibodies in both PrP(c) and PrP(sc). Four out of five of the anti-PrP antibodies raised in a prion knockout mouse immunized with the linear peptide of PrP90-120 could detect PrP(sc) in 'native' and denatured forms and PrP(c) in normal cells, as well as recognize epitopes within PrP93-112 residues. In contrast, the other six anti-PrP reagents, including five raised from the two knockout mice immunized with conformationally modified PrP90-120 peptide, could detect PrP(c) and recognize epitopes within PrP93-107 residues. Four of these reagents could also detect denatured PrP(sc) on western blots but not PrP(sc) plaques in brain tissue. The results indicate that residues PrP93-102 are exposed in PrP(c) but are buried upon conversion to the PrP(sc) isoform. Furthermore, PrP103-107 residues are partially buried in PrP(sc) while only the PrP107-112 epitope remains exposed, suggesting that the region PrP93-112 undergoes conformational changes during its conversion to PrP(sc).     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Rapid formation of amyloid from alpha-monomeric recombinant human PrP in vitro

The infectious agent of prion diseases is identified with PrP(Sc), a beta-rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrP(C). To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low-level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro kinetics of amyloid formation from recombinant human PrP(90-231) in vitro in the context of two common allelic forms of PrP found in human populations that are associated with differences in prion disease susceptibility and pathological phenotype. We show that human PrP amyloid forms readily from its PrP(C)-like state in vitro, that the lag time of the reaction can be further shortened by the presence of a "seed" of pre-formed PrP amyloid, and that amyloid propagation is more complex than a simple crystallization process. We further show that the kinetics of amyloid formation do not differ between the Met129 and Val129 allelomorphs of human PrP, and that amyloid from each functions as an equally effective seed in heterologous, as in homologous amyloid reactions. The results could illuminate the process of amyloid formation in vivo as well as help understanding prion pathogenesis.     

Conformation of PrP(C) on the cell surface as probed by antibodies

We have investigated the conformation of Syrian hamster PrP(C) on the surface of transfected CHO cells by performing cross-competition experiments between a set of nine monoclonal antibody fragments (Fab) directed to defined epitopes throughout the protein. No competition was observed between antibodies recognizing epitopes located within the unstructured N-terminal portion of PrP(C) and those recognizing epitopes located within the ordered C-terminal half of the molecule. However, competition was observed between antibodies recognizing overlapping epitopes and between antibodies recognizing epitopes lying adjacent to one another in the PrP sequence. Titrating the reactivity of each Fab against cell-surface PrP(C) revealed a clear heterogeneity in the accessibility of different specific epitopes. Fab D18, recognizing sequence incorporating the first alpha-helix of PrP(C), bound the largest fraction of the cell-surface PrP population. In contrast, Fab E123, binding an epitope at the extreme N terminus of PrP, and Fab 13A5, binding an epitope in the central region of PrP, were able to recognize fewer than half the number of PrP(C) molecules bound by Fab D18. The pattern of antibody reactivity we observed may, in part, result from N-terminal truncation of a proportion of PrP(C) molecules found at the cell surface. However, truncation cannot account for the marked disparity between exposure of the Fab D18 and 13A5 epitopes, which lie adjacent in the PrP sequence. The relative inaccessibility of the 13A5 epitope likely reflects either PrP(C)-PrP(C) interaction, interaction between PrP(C) and other constituents on the cell membrane, or the existence of PrP(C) subspecies with distinct conformations.     

Copper binding and conformation of the N-terminal octarepeats of the prion protein in the presence of DPC micelles as membrane mimetic

The prion protein is usually pictured as globular structured C-terminal domain that is linked to an extended flexible N-terminal tail. However, in its physiological form, it is a glycoprotein tethered to the cell surface via a C-terminal GPI anchor. The low solubility of PrP even without GPI anchor and its strong tendency for aggregation has forced most structural investigations to be performed at low pH and mostly with N-terminally truncated variants. In the present study, we have used a synthetic peptide related to the PrP tetra-octarepeat region, i.e., the sequence (Pro-His-Gly-Gly-Gly-Trp-Gly-Gln)(4), for NMR structural analysis of its preferred conformation in DPC micelles as membrane mimic. Well-defined and identical loops are observed between the four octarepeats that are linked by flexible Gly-Gly-Gly sequences. Interaction with the micelles is mainly through the tryptophan residues that appear to act as anchors. Copper binding to the peptide in the presence of DPC micelles revealed marked conformational rearrangements although binding to the micelles is preserved. Interestingly, titration experiments point to cooperative effects for the four binding sites. A destabilization of the DPC micelles by the peptide parallels the destabilizing effect of the prion protein on membranes so that the octarepeat region appears to be very membrane-active. How the physico-chemical properties reported here are linked to the function and significance of the prion protein remains a puzzle as long as the functional mechanism of the prion protein is not precisely elucidated. Nevertheless, our results emphasize the strong influence of the (membrane) environment on the PrP properties.     

Tau融合蛋白及其缺失突变体与朊蛋白的体外作用分析

在部分朊病毒病（prion diseases）中,高度磷酸化的微管相关蛋白tau与朊蛋白(prion protein,PrP)发生共定位,tau蛋白可能在朊病毒病的病理机制中有重要作用. 本室已经证明二者可以发生分子间相互作用,本文进一步分析了tau蛋白与prion的体外相互作用及作用位点. 利用RT-PCR方法从人源细胞系SHSY5Y cDNA中扩增出微管相关蛋白tau全长cDNA序列,克隆至质粒pGEX-2T载体,在大肠杆菌中诱导表达融合蛋白GST-tau. 利用GST pull-down及免疫共沉淀方法检测全长tau蛋白与PrP23-231的分子间相互作用. 进一步表达tau 蛋白的各种缺失突变体,确定tau蛋白与PrP蛋白的相互作用位点. 结果表明,所表达的全长tau蛋白及各种缺失突变体均为可溶性蛋白,Western印迹结果显示,各种蛋白均能很好的被tau蛋白单抗识别. GST pull-down和免疫共沉淀实验均显示,原核表达的全长tau蛋白可与全长的PrP蛋白在体外发生相互作用,并确定相互作用位点位于tau蛋白的N端序列及中段的重复区. 上述结果为研究tau蛋白与PrP的相互作用在朊病毒病的发病机制中的意义提供了一定的理论基础.     

Optimized overproduction,purification, characterization and high-pressure sensitivity of the prion protein in the native (PrP(C)-like) or amyloid (PrP(Sc)-like) conformation

Overproduction and purification of the prion protein is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. We have developed a method for the effective cloning, overexpression in Escherichia coli and purification to homogeneity of Syrian golden hamster prion protein (SHaPrP(90-231)). A high level of overexpression, resulting in the formation of inclusion bodies, was obtained under the control of the T7-inducible promoter of the pET15b plasmid. The protein required denaturation, reduction and refolding steps to become soluble and attain its native conformation. Purification was carried out by differential centrifugation, gel filtration and reverse phase chromatography. An improved cysteine oxidation protocol using oxidized glutathione under denaturing conditions, resulted in the recovery of a higher yield of chromatographically pure protein. About 10 mg of PrP protein per liter of bacterial culture was obtained. The recombinant protein was identified by monoclonal antibodies and its integrity was confirmed by electrospray mass spectrometry (ES/MS), whereas correct folding was assessed by circular dichroism (CD) spectroscopy. This protein had the structural characteristics of PrP(C) and could be converted to an amyloid structure sharing biophysical and biochemical properties of the pathologic form (PrP(Sc)). The sensitivity of these two forms to high pressure was investigated. We demonstrate the potential of using pressure as a thermodynamic parameter to rescue trapped aggregated prion conformations into a soluble state, and to explore new conformational coordinates of the prion protein conformational landscape.     

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).     

Toward molecular dissection of PrPC-PrPSc interactions

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.