Prion protein gene expression in cultured cells

A single copy gene encodes both the scrapie (PrP^Sc) and cellular(PrP^C) isoforms of the prion protein (PrP). Cultured cell lineswere found to express the endogenous PrP mRNA at levels comparableto those observed in the brains of adult rodents; however, thesecells were invariably found to express greatly reduced levelsof PrP. In all the cell lines examined, PrP was undetectableby Western immunoblot analysis. These cells were also poor recipientsfor expression constructs linking the hamster PrP gene openreading frame to several strong eukaryotic promoters; stableclones derived by transfection of these expression vectors failedto show elevated expression of PrP. When extremely high levelsof PrP mRNA were produced using either an insect baculovirusor a mammalian SV40 based vector, significant quantities ofPrP were produced, although in both cases the proteins wereapparently processed differently from the PrP^C observed in brains.In an expression system using an SV40 late promoter vector inmonkey COS-7 cells, a significant fraction of PrP was transportedto the cell surface where PrP^C is found in vivo. PrP synthesizedby the baculovirus vector failed to induce scrapie in hamstersand did not possess the characteristics of the PrP^Sc isoformassociated with infectivity. The SV40 late promoter vector systemmay permit experiments designed to elucidate the role of PrP^Scduring scrapie infection as well as the function of PrP^C innormal metabolism.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrP^Sc is the only identifiablecomponent of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakobdisease in humans are transmissible, degenerative neurologicaldiseases caused by prions. Standard predictive strategies havebeen used to analyze the secondary structure of the prion proteinin conjunction with Fourier analysis of the primary sequencehydrophobicities to detect potential amphipathic regions. Severalhydrophobic segments, a proline- and glycine-rich repeat regionand putative glycosylation sites are incorporated into a modelfor the integral membrane topology of PrP. The complete aminoacid sequences of the hamster, human and mouse prion proteinsare compared and the effects of residue substitutions upon thepredicted conformation of the polypeptide chain are discussed.While PrP has a unique primary structure, its predicted secondarystructure shares some interesting features with the serum amyloidA proteins. These proteins undergo a post-translational modificationto yield amyloid A, molecules that share with PrP the abilityto polymerize into birefringent filaments. Our analyses mayexplain some experimental observations on PrP, and suggest furtherstudies on the properties of the scrapie and cellular PrP isoforms.     

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrP^C) to convert into infectious prions (PrP^Sc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrP^C and PrP^Sc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrP^C to PrP^Sc conversion and determining PrP^C stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.     

Prion-Associated Neurodegeneration Causes Both Endoplasmic Reticulum Stress and Proteasome Impairment in a Murine Model of Spontaneous Disease

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrP^C to its abnormal and misfolded isoform PrP^Sc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrP^Sc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrP^Sc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the Ub^G76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the Ub^G76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.     

Strain Typing of Classical Scrapie and Bovine Spongiform Encephalopathy (BSE) by Using Ovine PrP (ARQ/ARQ) Overexpressing Transgenic Mice

Transmissible spongiform encephalopathies (TSE), caused by abnormal prion protein (PrP^Sc), affect many species. The most classical scrapie isolates harbor mixtures of strains in different proportions. While the characterization of isolates has evolved from using wild-type mice to transgenic mice, no standardization is established yet. Here, we investigated the incubation period, lesion profile and PrP^Sc profile induced by well-defined sheep scrapie isolates, bovine spongiform encephalopathy (BSE) and ovine BSE after intracerebral inoculation into two lines of ovine PrP (both ARQ/ARQ) overexpressing transgenic mice (Tgshp IX and Tgshp XI). All isolates were transmitted to both mouse models with an attack rate of almost 100%, but genotype-dependent differences became obvious between the ARQ and VRQ isolates. Surprisingly, BSE induced a much longer incubation period in Tgshp XI compared to Tgshp IX. In contrast to the histopathological lesion profiles, the immunohistochemical PrP^Sc profiles revealed discriminating patterns in certain brain regions in both models with clear differentiation of both BSE isolates from scrapie. These data provide the basis for the use of Tgshp IX and XI mice in the characterization of TSE isolates. Furthermore, the results enable a deeper appreciation of TSE strain diversity using ovine PrP overexpressing transgenic mice as a biological prion strain typing approach.     

Structural Mechanism of Barriers to Interspecies Seeding Transmissibility of Full-Length Prion Protein Amyloid

A puzzling feature of prion diseases is the cross-species barriers. The detailed molecular mechanisms underlying these interspecies barriers remain poorly understood because of a lack of high-resolution structural information on the scrapie isoform of the prion protein (PrP^Sc). In this study we identified the critical role of the residues 165/167 in the barrier to seeding mouse PrP (mPrP) fibril seeds to human cellular prion protein (PrP^C). Solid-state NMR revealed a C-terminal β-sheet core spanning residues 165–230 and the packing arrangement of mPrP fibrils. Residues 165/167 are located on one end of the fibril core. Molecular dynamics simulations demonstrated that the stabilities of the seeding-induced β-strand structures are significantly impacted by hydrogen bonds involving the side chain of residue 167 and steric resistance involving residue 165. These findings suggest that the α2–β2 loop containing residues 165/167 could be the initial site of seed–template conformational conversion.     

Ethanolamine Is a New Anti-Prion Compound

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrP^Sc. PrP^Sc is produced through conformational conversion of the cellular isoform of prion protein, PrP^C, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrP^Sc levels when cultured in advanced Dulbecco’s modified eagle medium (DMEM) but not in classic DMEM. PrP^C levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrP^Sc levels in N2aC24L1-3 cells, but not PrP^C levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.     

Discovery of a Class of Diketopiperazines as Antiprion Compounds

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimer's have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d , which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism‐of‐action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrP^Sc)‐like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.     

Selection of novel ligands from a whole-molecule randomly mutated C5a library

Novel antagonists of the proinflammatory leukocyte chemoattractantC5a have been produced from a phage display library of whole-moleculerandom mutants. The cDNA for the inflammatory polypeptide C5adR⁷⁴was used as template in a PCR reaction doped with the mutagenicnucleoside triphosphates dPTP {dP: 6-(2-deoxy-ß-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one}and 8-oxodGTP (8-oxodG: 8-oxo-2'-deoxyguanosine) to allow theintroduction of mutations in a highly controlled manner throughoutthe cDNA. The resultant library of mutants was displayed onbacteriophage M13 using a jun/fos linker sequence. Functionalpolypeptides were isolated by several rounds of selection againstthe receptor for C5a expressed on the surface of CHO cells.From this selection procedure, a limited number of variantsof C5adR⁷⁴ were obtained. When expressed as free polypeptide,the binding affinities of the selected C5adR⁷⁴ sequences wereincreased 5-fold relative to wild-type protein. Site-directedmutagenesis of the C-terminus of these variants resulted inthe production of antagonists of C5adR⁷⁴ activity.     

Distinctive Toll-like Receptors Gene Expression and Glial Response in Different Brain Regions of Natural Scrapie

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumulation of disease-specific prion protein (PrP^Sc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion diseases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed the gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated the possible correlations between gene expression and the pathological hallmarks of prion disease. We observed significant changes in TLR expression in scrapie-infected sheep that correlate with the degree of spongiosis, PrP^Sc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, regardless of the severity of neuropathology. In the hippocampus, we observed milder neuropathology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, a comparison of the findings in naturallyinfected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLRgene expression.     

The de novo protein with grafted biological function: transferring of interferon blast-transforming activity to albebetin

The de novo protein albebetin has been designed recently toform a predetermined tertiary fold that has not yet been observedin natural proteins. An eight amino acid fragment (131–138)of human interferon ₂ carrying the blasttransforming activityof the protein was attached to the N-terminus of albebetin nextto its initiatory methionine residue. The gene of chimeric proteinwas expressed in a wheat germ cell-free translation system andsynthesized protein was tested for its compactness and stability.Its ability for receptor binding was also studied. We have shownthat albebetin with attached octapeptide is practically as compactas natural proteins of corresponding molecular weight and possesseshigh stability toward the urea-induced unfolding. It binds murinethymocyte receptor at a high affinity and activates the thymocyteblast transformation efficiently at a concentration of 10^-11M.     

The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c

The gene encoding trypsin-solubilized bovine liver microsomalcytochrome b₅ (82 residues in length) has been mutated, in whichthe codons of Glu44 and Glu56 were changed to those of Ala.The mutated genes were expressed in Escherichia coli successfullyand three mutant proteins (E44A, E56A and E44/56A) were obtained.The UV-visible, CD and ¹H NMR spectra of proteins have beenstudied. The results show that the mutagenesis at surface residuesdoes not alter the secondary and tertiary structures of cytochromeb₅ significantly. The interactions between recombinant cytochromeb₅ and its mutants with cytochrome c were studied by using opticaldifference spectra. The results demonstrated that both Glu44and Glu56 of cytochrome b₅ participate in the formation of acomplex between cytochrome b₅ and cytochrome c.     

High-level bacterial expression, purification and characterization of human calreticulin

To investigate its cellular function and role in autoimmunedisease pathogenesis, we have bacterlally expressed human calreticulin,a major calcium-binding protein in the endoplasmic reticulumand a human autoantigen. This is the First report describingthe heterologous expression of calreticulin from any source.The recombinant calreticulin constituted {small tilde}32% ofthe soluble Escherichia coli proteins, and was purified to apparenthomogeneity by ion exchange and hydrophobic liquid chromatography.As does the bona fide protein, the recombinant calreticulinbinds calcium and undergoes changes in its conformation uponZn²⁺ binding. We take this as a strong indication that the foldingof the E.coli-expressed calreticulin is very similar, if notidentical, to that of the authentic protein. Moreover, the bacteriallyexpressed calreticulin readily reacted with anti-human and anti-rabbitantibodies, and the anti-recombinant calreticulin antibodiesimmunoreacted with HeLa calreticulin. The availability of thisexpression system will allow us to carry out site-specific anddeletion mutagenesis analysis in structure-function studiesof calreticulin.     

Prion-protein-specific aptamer reduces PrPSc formation

The critical initial event in the pathophysiology of transmissible spongiform encephalopathies (TSEs) appears to be the conversion of the cellular prion protein (PrP(C)) into the abnormal isoform PrP(Sc). This isoform forms high-molecular-weight protease K (PK) resistant aggregates that accumulate in the central nervous system of affected individuals. We have selected nuclease-resistant 2'-amino-2'-deoxypyrimidine-modified RNA aptamers which recognize a peptide comprising amino acid residues 90-129 of the human prion protein with high specificity. This domain of prion proteins is thought to be functionally important for the conversion of PrP(C) into its pathogenic isoform PrP(Sc) and is highly homologous among prion proteins of various species including mouse, hamster, and man. Consequently, aptamer DP7 binds to the full-length human, mouse, and hamster prion protein. At low concentrations in the growth medium of persistently prion-infected neuroblastoma cells, aptamer DP7 significantly reduced the relative proportion of de novo synthesized PK-resistant PrP(Sc) within only 16 h. These findings may open the door towards a rational development of a new class of drugs for the therapy or prophylaxis of prion diseases.     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA^1–413.DHFR^1–187.ETA^609–613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes.     

Protein engineering of cytochrome c by semisynthesis: substitutions at Glutamic acid 66

We have used protein semisynthesis to prepare four analoguesof horse cytochrome c, in which the glutamic acid residue atposition 66 has been removed and replaced by norvaline, glutamine,lysine and, as a methodological control, glutamic acid. Thisresidue is quite strongly conserved in mitochondrial cytochromec, and forms part of a cluster of acidic residues that occursin all cytochromes c but whose function is obscure. Comparativestudies of the physical and biochemical properties of the analogueshave now disclosed two specific roles for Glu⁶⁶ in the protein.It contributes significantly to the stabilization of the activeconformation of the protein, probably by salt bridge formation,and it appears to participate in the redox-state-dependent ATP-bindingsite of cytochrome c. Our results also support two general viewsof the role of surface charged residues in cytochrome c, namelythat their disposition influences both redox potential, throughthe electrostatic field felt at the redox centre, and the kineticsof electron transfer, through the dipole moment they generate.     

Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C- terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain.      

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.