Species barriers in a model for specific prion protein dimerisation

It has been proposed that the most highly conserved sequence segment within the prion protein (PrP) may be involved in dimer formation within both the normal (PrPC) and misfolded (PrPSc) forms. This hypothesis is now examined in the context of amino acids known to be involved in species barriers or in disease modifying polymorphisms, and the structure of a mouse PrP fragment. These locations can be plausibly explained on the basis of the specific dimer model, so that a potential role for a conserved dimerisation element in prion disease progression cannot be excluded.     

Proof of the primacy of prion protein

This paper presents a case of a 5-year old girl with a congenital absence of the nose. Congenital arrhinia is a very rare malformation of the midfacial bones. The difficulties of treating a child with this abnormality are discussed.     

Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations

We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies.     

A trend of molecular genetics on prion diseases and prion protein

Infectious amyloid filaments designated as prion rods or scrapie associated fibrils (SAF) present in brain tissues affected by transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker disease (GSS) and kuru of humans, and scrapie of sheep. A hydrophobic glycoprotein, PrPSc is a major component of SAF, and is known to be associated with the infectivity of these diseases. Both PrPSc and the normal isoform of this glycoprotein, PrPC are encoded by a single host gene, PrP gene, and the conversion of PrPC to PrPSc is a posttranslational event. Several mutations on the PrP gene are associated with variations of the phenotype and the occurrence in familial CJD and GSS.     

Mapping the prion protein using recombinant antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPC but absent from PrPSc.     

Doxycycline control of prion protein transgene expression modulates prion disease in mice

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at approximately 50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.     

A prion disease with a novel 96-base pair insertional mutation in the prion protein gene

There are coding mutations in the prion protein gene in familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and other phenotypes that make up the inherited prion diseases. Insertional mutations consisting of two, five, six, seven, eight, and nine additional octapeptide repeat elements are seen in the inherited prion diseases and usually present as atypical dementias with considerable intrafamilial phenotypic variability. A four-octarepeat insertion was reported previously in an individual without neurodegenerative disease who died of hepatic cirrhosis. Here we report a novel four-octarepeat insertional mutation in a case with classical clinical, electroencephalographic and histopathologic features of CJD with the unusual finding of pronounced prion protein immunoreactivity of the molecular layer of the cerebellum.     

Solid-state NMR studies of the prion protein H1 fragment

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

Astrocytic glutamate uptake and prion protein expression

Factors influencing glutamate uptake by astrocytes may indirectly influence neuronal survival. Elevated extracellular glutamate may be excitotoxic or may exacerbate neurodegeneration in various neurological diseases. By using a cell culture model, we have investigated the influence of astrocytic prion protein (PrPc) expression on glutamate uptake. Type 1 astrocytes expressing PrPc have a higher rate of Na+-dependent glutamate uptake than PrPc-deficient type 1 astrocytes. This difference is exacerbated when serum free media is used to culture the astrocytes. Further analysis suggested that a decrease in substrate affinity is responsible for the sensitivity of PrP-deficient astrocytic glutamate uptake to culture conditions. PrPc has been shown to bind copper. Greater sensitivity of cells to copper concentrations may be responsible for the decreased substrate affinity observed. PrPc-deficient cerebellar cells are more sensitive to glutamate toxicity in the presence of copper. These results show that glutamate uptake from astrocytes is dependent on PrPc expression which in turn may be related to copper metabolism.     

Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes

Mammalian cells contain at least three signaling systems which are structurally related to the mitogen-activated protein kinase (MAPK) pathway. Growth factors acting through Ras primarily stimulate the Raf/MEK/MAPK cascade of protein kinases. In contrast, many stress-related signals such as heat shock, inflammatory cytokines, and hyperosmolarity induce the MEKK/SEK(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38(hog) pathways. Physiological agonists of these pathway types are either qualitatively or quantitatively distinct, suggesting few common proximal signaling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the components of all three pathways. These studies suggest a high degree of cross-talk apparently not seen in vivo. We have examined the possible molecular basis of the differing agonist profiles of these three MAPK pathways. We report preferential association between MAP kinases and their activators in eukaryotic cells. Furthermore, using the yeast 2-hybrid system, we show that association between these components can occur independent of additional eukaryotic proteins. We show that SAPK(JNK) or p38(hog) activation is specifically impaired by co-expression of cognate dominant negative MAP kinase kinase mutants, demonstrating functional specificity at this level. Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activation of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38(hog) phosphorylation. Therefore, in intact cells, the three MAPK pathways may be independently regulated and their components show specificity in their interaction with cognate cascade members. The degree of intermolecular specificity suggests that mammalian MAPK signaling pathways may remain distinct without the need for specific scaffolding proteins to sequester components of individual pathways.     

Impaired motor coordination in mice lacking prion protein

1. Prion protein (PrPC) is a host-encoded glycoprotein constitutively expressed on the neuronal cell surface. Accumulation of its protease-resistant isoform is closely related to pathologic changes and prion propagation in the brain tissue of a series of prion diseases. However, the physiological role of PrPC remains to be elucidated. 2. After long-term observation, we noted impaired motor coordination and loss of cerebellar Purkinje cells in the aged mice homozygous for a disrupted PrP gene, a finding which strongly suggests that PrPC plays a role in the long-term survival of Purkinje cells. 3. We also describe the resistance of the PrP null mice to the prion, indicating the requirement of PrPC for both the development of prion diseases and the prion propagation.     

Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-sheet. The "protein-only" hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.     

Strain-dependent differences in beta-sheet conformations of abnormal prion protein

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.     

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.     

Chicken monoclonal antibodies against synthetic bovine prion protein peptide

A night course taken almost 25 years ago sparked an interest in Leonardo da Vinci that has become a passion for a London, Ont., neurosurgeon. Dr. Rolando Del Maestro now boasts one of the largest collections of da Vinci artifacts in North America.     

Propagation of Long Water Waves through Branching Channels

This paper investigates the fundamental behavior of long water waves propagating through branching channels of uniform depth and width. Numerical simulations based on the Boussinesq long wave model were carried out to investigate the effects of channel width b, effective wavelength λe, wave amplitude α, and angle θ between channel branches on wave transmission and reflection. Our results showed that the transmission and reflection of long waves through branching channels are dominantly governed by a single dimensionless parameter b/λe, whereas other parameters such as the angle between channel branches are less important. Detailed quantitative results for predicting long wave transmission and reflection in different branching channels based on the similarity parameter b/λe were determined. From these results, we have discovered some very interesting and distinct long wave behaviors in narrow and wide branching channels. To verify the numerical results, experiments were conducted in a right-angled branching channel, and the experimental results showed good agreement with the numerical predictions.     

Expression of cellular prion protein in activated hepatic stellate cells

OBJECTIVES: We tested the hypothesis that, in patients with stable heart failure, measuring big endothelin-1 (ET-1) plasma level at rest predicts short-term prognosis better than peak oxygen consumption (VO2max) at exercise. BACKGROUND: Cardiopulmonary exercise testing and evaluation of neurohumoral plasma factors are established tools to estimate survival in patients with heart failure. No data, however, exist comparing the prognostic value of both marker categories simultaneously. METHODS: Two hundred twenty-six heart failure patients were studied in regard to a combined end point of death and prioritization for urgent cardiac transplantation within 1 year follow-up. RESULTS: During the study period 149 patients were without cardiac events (group A), 69 patients died or were urgently transplanted (group B) and 8 patients were alive after a nonurgent heart transplant operation. Norepinephrine (p < 0.0001), atrial natriuretic peptide (p < 0.001), big endothelin plasma levels (p < 0.0001 as well as workload, VO2max and achieved percentage of predicted peak oxygen consumption (pVO2max) (all p < 0.0001) differed significantly between groups A and B. In multivariate stepwise regression analysis, however, only big ET-1 plasma concentration (chi2=74.4, p < 0.0001), New York Heart Association function class (chi2=33.9, p < 0.0001), maximal workload (chi2=7.2, p < 0.01, and plasma atrial natriuretic peptide (ANP) concentration (chi2=4.6, p < 0.05) were independently related to outcome. Peak oxygen consumption or pVO2max did not reach statistical significance in this model. Event-free survival rates were significantly lower in patients with a big ET-1 level of 4.3 fmol/ml or more than with lower big ET-1 levels (p < 0.0001). CONCLUSION: We conclude that in patients with chronic heart failure who are stable on oral therapy measuring big ET-1 and ANP plasma levels may be a valuable noninvasive adjunct to improve the prognostic accuracy of detecting high risk patients compared with exercise testing alone.     

Recombinant scrapie-like prion protein of 106 amino acids is soluble

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.