Evidence for oxidative damage to prion protein in prion diseases

In prion diseases the irreversible protein structural transformation process is completed in the brains of mammals within a few months, the uniformly generated infectivity displays extraordinary resistance to inactivation, suggesting that a vital energy source is required for the production of infectious particles. Considering the high oxygen-respiration rate in the brains, prion protein oxidative damage can be the crucial factor. Both theoretical consideration of the nature of protein radical reactions and a large body of previously unraveled feature of scrapie and prion diseases have provided multiple distinct lines of compelling evidence which persuasively support a suggestion that the infectious agents may be prion (free) radicals produced from protein oxidative damage. This paper describes that scrapie prions are most likely formed from prion radicals and oxidative species-mediated sequence-specific cross-linking of benign prion proteins.     

Antibodies to a scrapie prion protein

Scrapie is a slow infection of the nervous system which progresses in the absence of any apparent immune response. The recent development of a large-scale purification protocol for scrapie prions made it possible to obtain substantial quantities of electrophoretically purified prion protein (PrP 27-30) and we report here on the successful production of a rabbit antiserum to PrP 27-30. The antiserum reacted with PrP 27-30 and several lower molecular weight proteins as shown by Western blots; it did not react with protein preparations from uninfected brains. Discrete structures in the subependymal region of scrapie-infected hamster brains were stained immunocytochemically. These same structures also stained with Congo red dye and showed green birefringence with polarized light, a characteristic of purified prion rods. This staining pattern suggests that they are amyloid plaques.     

Binding of disease-associated prion protein to plasminogen

Transmissible spongiform encephalopathies are associated with accumulation of PrP(Sc), a conformer of a cellular protein called PrP(C). PrP(Sc) is thought to replicate by imparting its conformation onto PrP(C) (ref. 1), yet conformational discrimination between PrP(C) and PrP(Sc) has remained elusive. Because deposition of PrP(Sc) alone is not enough to cause neuropathology, PrP(Sc) probably damages the brain by interacting with other cellular constituents. Here we find activities in human and mouse blood which bind PrP(Sc) and prion infectivity, but not PrP(C). We identify plasminogen, a pro-protease implicated in neuronal excitotoxicity, as a PrP(Sc)-binding protein. Binding is abolished if the conformation of PrP(Sc) is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I-III) retains this binding activity, and binding can be competed for with lysine. Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.     

Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.

Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrP(Sc) replicate by conversion of host PrP(C), generating large amounts of PrP(Sc) aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrP(C) into a protease-resistant, PrP(Sc)-like form in the presence of minute quantities of PrP(Sc) template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrP(Sc) is incubated with PrP(C) are disrupted by sonication to generate multiple smaller units for the continued formation of new PrP(Sc). After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrP(Sc) replication results in the generation of infectivity in vitro.     

Linkage of a prion protein missense variant to Gerstmann-Str?ussler syndrome

Gerstmann-Str?ussler syndrome is a rare familial neurodegenerative condition that is vertically transmitted, in an apparently autosomal dominant way. It can also be horizontally transmitted to non-human primates and rodents through intracerebral inoculation of brain homogenates from patients with the disease. The exact incidence of the syndrome is unknown but is estimated to be between one and ten per hundred million. Patients initially suffer from ataxia or dementia and deteriorate until they die, in one to ten years. Protease-resistant prion protein (PrP) and PrP-immunoreactive amyloid plaques with characteristic morphology accumulate in the brains of these patients. Current diagnostic criteria for Gerstmann-Str?ussler syndrome incorporate clinical and neuropathological features, as animal transmission studies can be unreliable. PrP is implicated in the pathogenesis and transmission of the condition and in scrapie, an equivalent animal disease. It was discovered by enriching scrapie-infected hamster brain fractions for infectivity. Because there is compelling evidence that the scrapie isoform of PrP is a necessary component of the infectious particle, it seemed possible that the PrP gene on the short arm of human chromosome 20 in Gerstmann-Str?ussler syndrome might be abnormal. We show here that PrP codon 102 is linked to the putative gene for the syndrome in two pedigrees, providing the best evidence to date that this familial condition is inherited despite also being infectious, and that substitution of leucine for proline at PrP codon 102 may lead to the development of Gerstmann-Str?ussler syndrome.     

Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.

The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss by deletion, mutation or epigenetic silencing is among the most frequently observed molecular lesions in human cancer. Overlapping reading frames in the INK4A/ARF gene encode p16INK4a and a distinct tumour-suppressor protein, p19ARF (ref. 3). Here we describe the generation and characterization of a p16Ink4a-specific knockout mouse that retains normal p19Arf function. Mice lacking p16Ink4a were born with the expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16Ink4a exhibited enhanced mitogenic responsiveness, consistent with the established role of p16Ink4a in constraining cellular proliferation. In contrast to mouse embryo fibroblasts (MEFs) deficient in p19Arf (ref. 4), p16Ink4a-null MEFs possessed normal growth characteristics and remained susceptible to Ras-induced senescence. Compared with wild-type MEFs, p16Ink4a-null MEFs exhibited an increased rate of immortalization, although this rate was less than that observed previously for cells null for Ink4a/Arf, p19Arf or p53 (refs 4, 5). Furthermore, p16Ink4a deficiency was associated with an increased incidence of spontaneous and carcinogen-induced cancers. These data establish that p16Ink4a, along with p19Arf, functions as a tumour suppressor in mice.     

A new approach to protein fold recognition.

The prediction of protein tertiary structure from sequence using molecular energy calculations has not yet been successful; an alternative strategy of recognizing known motifs or folds in sequences looks more promising. We present here a new approach to fold recognition, whereby sequences are fitted directly onto the backbone coordinates of known protein structures. Our method for protein fold recognition involves automatic modelling of protein structures using a given sequence, and is based on the frameworks of known protein folds. The plausibility of each model, and hence the degree of compatibility between the sequence and the proposed structure, is evaluated by means of a set of empirical potentials derived from proteins of known structure. The novel aspect of our approach is that the matching of sequences to backbone coordinates is performed in full three-dimensional space, incorporating specific pair interactions explicitly.     

The Lyme disease agent exploits a tick protein to infect the mammalian host

The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick-mouse cycle. Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.     

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.     

Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.

A locus segregating with familial Alzheimer's disease (AD) has been mapped to chromosome 21, close to the amyloid precursor protein (APP) gene. Recombinants between the APP gene and the AD locus have been reported which seemed to exclude it as the site of the mutation causing familial AD. But recent genetic analysis of a large number of AD families has demonstrated that the disease is heterogeneous. Families with late-onset AD do not show linkage to chromosome 21 markers. Some families with early-onset AD show linkage to chromosome 21 markers, but some do not. This has led to the suggestion that there is non-allelic genetic heterogeneity even within early onset familial AD. To avoid the problems that heterogeneity poses for genetic analysis, we have examined the cosegregation of AD and markers along the long arm of chromosome 21 in a single family with AD confirmed by autopsy. Here we demonstrate that in this kindred, which shows linkage to chromosome 21 markers, there is a point mutation in the APP gene. This mutation causes an amino-acid substitution (Val----Ile) close to the carboxy terminus of the beta-amyloid peptide. Screening other cases of familial AD revealed a second unrelated family in which this variant occurs. This suggests that some cases of AD could be caused by mutations in the APP gene.