Proteinaceous Infectious Agents Research Articles

High-resolution structure of infectious prion protein: the final frontier

Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrP(Sc)), which is able to template the conversion of the host's natively folded form of the protein (PrP(C)). The detailed mechanism of prion replication and the high-resolution structure of PrP(Sc) are unknown. The currently available information on PrP(Sc) structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrP(Sc) structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrP(Sc), arguably the major pending milestone to reach in understanding prions.

The prion hypothesis: from biological anomaly to basic regulatory mechanism.

Prions are unusual proteinaceous infectious agents that are typically associated with a class of fatal degenerative diseases of the mammalian brain. However, the discovery of fungal prions, which are not associated with disease, suggests that we must now consider the effect of these factors on basic cellular physiology in a different light. Fungal prions are epigenetic determinants that can alter a range of cellular processes, including metabolism and gene expression pathways, and these changes can lead to a range of prion-associated phenotypes. The mechanistic similarities between prion propagation in mammals and fungi suggest that prions are not a biological anomaly but instead could be a newly appreciated and perhaps ubiquitous regulatory mechanism.

Estimating prion concentration in fluids and tissues by quantitative PMCA

Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrP(Sc)) in brain, spleen, blood and urine of scrapie-affected hamsters.

De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype

Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein (PrPSc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein (PrPC). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrPSc particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrPSc in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrPSc by the protein misfolding cyclic amplification (PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrPSc was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrPSc in the absence of pre-existing PrPSc in different animal species at a low and variable rate. De novo generated PrPSc was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.

Photocatalytic degradation of prions using the photo-Fenton reagent

Prions are proteinaceous infectious agents postulated to be the causative agents of a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). A known iatrogenic transmission route of TSEs to humans occurs via prion-contaminated surgical instruments or biological materials. Prions, unlike most common pathogens, exhibit an extraordinary resistance to conventional decontamination procedures. We have recently demonstrated that the application of TiO(2)-based heterogeneous photocatalytic oxidation is able to significantly reduce prion infectivity. The present study investigates the potential of a homogeneous photocatalytic method, based on the photo-Fenton reagent, to degrade prion proteins. We show that the photo-Fenton reagent efficiently degrades not only recombinant prion proteins, but also the total protein amount from brain preparations of naturally or experimentally infected species and PrP(Sc) (PrP scrapie) contained in sheep scrapie brain homogenates.

Presymptomatic Detection of Prions in Blood

Prions are thought to be the proteinaceous infectious agents responsible for transmissible spongiform encephalopathies (TSEs). PrP(Sc), the main component of the infectious agent, is also the only validated surrogate marker for the disease, and its sensitive detection is critical for minimizing the spread of the disease. We detected PrP(Sc) biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrP(Sc) detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrP(Sc) in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs.

High Affinity Binding between Copper and Full-length Prion Protein Identified by Two Different Techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.

Diagnosing prion diseases: needs, challenges and hopes.

Prion diseases are among the most intriguing infectious diseases and are associated with unconventional proteinaceous infectious agents known as prions. Prions seem to lack nucleic acid and propagate by transmission of protein misfolding. The nature of prions and their unique mode of transmission present challenges for early diagnosis of prion diseases. In this article, state-of-the-art prion diagnostic techniques, together with the new strategies that are being used to develop sensitive, early and non-invasive diagnoses for these diseases are reviewed.

Detergency of stainless steel surface soiled with human brain homogenate: an XPS study

In the detergency field of re-usable medical devices, a special attention is focused on the non conventional transmissible agent called prions which is a proteinaceous infectious agent. Few cleaning procedures are effective against prions and few techniques are available to study cleaning effectiveness with respect to proteins in general. In our study, X-ray photoelectron spectroscopy (XPS) has been used to evaluate the effectiveness of detergent formulations to remove proteins from stainless steel surface soiled with a brain homogenate (BH) from human origin. Our results showed that XPS is a reliable surface analysis technique to study chemical species remaining on surface and substrate properties after cleaning procedures. A semi-quantitative evaluation of the detergency effectiveness could also be performed.

The challenge of prion decontamination.

Prions are a class of proposed proteinaceous infectious agents that cause similar fatal brain diseases. These diseases are known as transmissible spongiform encephalopathies (TSEs), and include Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler syndrome (GSS) in humans, bovine spongiform encephalopathy (BSE) in cattle (also variant CJD [vCJD] in humans), and scrapie in sheep. latrogenic transmission of these diseases via contaminated medical devices is considered rare, but it has been documented. In an experimental case, transmission of the disease was observed despite cleaning and decontamination of the medical device with formaldehyde [1]. Clinically, 2 cases of TSEs have been confirmed in which transmission of the disease was due to the use of implanted depth electrodes that had been previously used on a patient with CJD and were inadequately decontaminated by cleaning with benzene and disinfecting with 70% alcohol and formaldehyde [2]. Other cases have been linked to the use of neurosurgical instruments [3]. These reports highlight the need for safe and effective prion decontamination methods. Recommended prion decontamination

Potential for transmission of prion disease by contact lenses: an assessment of risk.

Prions are small proteinaceous infectious agents known to cause central nervous system infections in both animals and humans. Interest in the pathogenesis of these diseases has grown since the emergence of bovine spongiform encephalopathy (BSE or "mad cow disease") in the United Kingdom and several other countries in Europe. Ingestion of meat products from animals infected with BSE has resulted in transmission of the disease to humans as a variant form of Creutzfeldt-Jakob Disease (vCJD). CJD has a long asymptomatic incubation period, is untreatable, and universally fatal. Hence concern has arisen over other possible routes of disease transmission. Because it is known that prions are found in low levels in the corneas of animals with experimentally induced prion disease, and that a case of human CJD transmission by corneal transplantation has occurred, the question of possible prion transmission through the reuse of diagnostic fitting of contact lenses has surfaced. This article reviews prion diseases of animals and humans, and the data regarding the presence and location of prions in the eye. Issues inherent in the question of corneal and contact lens transmission are discussed. Although some key information has yet to be derived, it appears that the chance of obtaining prion disease through contact lens use is negligible.

Transmission of spongiform encephalopathies (prion diseases)

The transmissible spongiform encephalopathies (TSE), or prion diseases, constitute a form of degenerative disorders of the central nervous system, which are characterized by a typical spongiform histological pattern and a fatal course. According to Prusiner's theory, its agent consists of a protein without any nucleic acid, the "proteinaceous infectious agent", or prion. This is a pathologically folded form of the normal prion protein (PrPC), and then called PrPSc. TSE are observed in different mammals including humans. In humans, they include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), Kuru and the new variant of the Creutzfeldt-Jakob disease (vCJD). The longest known TSE is scrapie in sheep. Since 1984 the bovine spongiform encephalopathy (BSE) and since 1996 the variant Creutzfeldt-Jakob disease have been documented. This paper gives an overview on the pathogenesis, epidemiology and the clinical features of the different prion diseases.

Prion disease and medical devices.

Prions are novel proteinaceous-infectious agents that have been implicated in transmissible spongiform encephalopathies. It is now generally accepted that noninfectious prion proteins are normally produced by the host and may undergo a conformational change to an abnormal, pathologic form, which appears to be responsible for disease symptoms. Many methods of decontamination and sterilization are claimed to be ineffective against prion proteins. Incidences of iatrogenic transmission of prions due to medical devices have been reported, and the recommended clinical practices when handling suspected cases are reviewed. Recent results with a peracetic acid based sterilant indicate that it may be a safe and effective means of prion inactivation on medical devices.

Elevated levels of tau-protein in cerebrospinal fluid of patients with Creutzfeldt–Jakob disease

Creutzfeldt–Jakob disease (CJD) is a rare, fatal, neurodegenerative disease caused by a transmissible agent designated as proteinaceous infectious agent (prion). Searching for biochemical markers of CJD, we analysed cerebrospinal fluid (CSF) samples of 53 patients for tau-protein using an enzyme linked immunoassay (ELISA). In a group of 21 patients with definite CJD seen in the German case control study for CJD, tau-protein concentrations in CSF were significantly higher than in two control-groups of patients with other diseases (median 13153 pg/ml, range 1533–27648 pg/ml; P=0.0001). One control group comprised 19 patients who were seen in the same study and were diagnosed as having other dementing diseases (tau concentration: median 558 pg/ml, range 233–1769 pg/ml). The second control group comprised 13 patients from our hospital with no dementing disease (tau concentration: median 296 pg/ml, range 109–640 pg/ml). We conclude that determination of tau protein levels in CSF is a useful marker for laboratory diagnosis of CJD.

The spectrum of transmissible spongiform encephalopathies.

Since the first description by A.M. Jakob and H.G. Creutzfeldt, five human diseases have been identified as transmissible spongiform encephalopathies (TSE). The disease bearing these authors' name, Creutzfeldt-Jakob disease (CJD) occurs sporadically, may be transmitted and has a genetic basis in 10-15% of all cases. Genetic diseases are the Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. The latest form of CJD in humans, variant CJD (vCJD), was first described in 1996 and may be considered as evidence for a link between human TSEs and those in the animal kingdom. The putative agent of all TSEs is a proteinaceous infectious agent or prion. The gene for the physiological isoform of this protein (prion related protein or PrPc)-is encoded on the short arm of chromosome 20 in humans. The role of the physiological isoform of PrPc is unknown. The physiological isoform PrPc is protease-sensitive and thus designated as PrP-sen, while the pathological isoform is protease-resistant and thus called PrP-res. The pathological isoform PrP-res is invariably associated with TSEs and has given rise to the term prion diseases. PrP-sen and PrP-res have an identical amino acid sequence and have identical posttranslational modifications as assessed by currently available methodology but differ in their tertiary and quaternary structure. These structural differences are thought to be propagated by interaction of PrP-res with PrP-sen, appear to be governed by the polymorphism of PrPc and have been shown to be invariably associated with disease. PrP-sen has a high content of alpha-helical structures, PrP-res consists predominantly of beta-pleated sheets. These physicochemical characteristics of PrP-res cause it to behave like amyloid. The neuropathological hallmarks of TSEs or prion diseases are spongiform change, astrocytic gliosis, neuronal loss and PrP-positive plaques. A nucleic acid has never consistently been shown in any TSE. Diagnosis of TSE can be reliably made only postmortem.

Diagnosis of Creutzfeldt-Jakob disease and related human spongiform encephalopathies

Spongiform encephalopathies are transmissible diseases (TSE) of animals and humans. With the appearance of bovine spongiform encephalopathy (BSE) in 1986 and in 1996 with the identification of an apparently new variant of the human spongiform encephalopathy Creutzfeldt-Jakob disease (CJD), great concerns of a potential transmission of BSE to humans have been voiced. The agent known to transmit CJD and other human and animal spongiform encephalopathies is designated as prion, i.e., proteinaceous infectious agent, due to the absence of evidence for the involvement of a nucleic acid in disease transmission. In humans the clinical diagnosis of typical CJD cases can now be supported by paraclinical parameters. Electroencephalographic changes, so called periodic sharp wave complexes, are pathognomonic for CJD but by no means specific. The detection of neuronal enzymes in the cerebrospinal fluid (CSF) such as neuron specific enolase (NSE) or glial proteins such as S-100 aids greatly in the diagnosis of a human spongiform encephalopathy. By far the most specific marker in CSF are a group of proteins designated 14-3-3. Current evidence suggests that by including elevated levels of NSE (> or = 35 ng/mL), S-100 (> or = 8 ng/mL) and tau protein in the CSF and the presence of 14-3-3, a laboratory supported diagnosis of CJD can be achieved which in the appropriate clinical setting has a better diagnostic accuracy than the currently used clinical and paraclinical diagnostic criteria alone.

Creutzfeld-Jakob disease: Recommendations for infection control

Creutzfeld-Jakob disease: an infectious, progressive, degenerative neurologic disorder, has a presumably long incubation period but a rapid, fatal course. Brain tissue at autopsy resembles that seen in spongioform encephalopathies of other species. Creutzfeld-Jakob disease is transmitted by a proteinaceous infectious agent, or “prion.” Epidemiologic patterns remain uncertain; various studies have reported conflicting risk factors in different populations, and genetic susceptibility may be involved. Although natural transmission routes are still unclear, both iatrogenic and nosocomial transmissions have been identified. Transmission has occurred through contaminated electrodes, contaminated biologic products from cadaveric brains, and infected donor tissues, including dura mater and corneas. Because the prion is difficult to eradicate, stringent sterilization precautions must be taken with all surgical instruments. Some tissues and body fluids (e.g., brain, ocular, central nervous system) from the patient with Creutzfeld-Jakob disease are highly infectious and must be contained or incinerated. Some body fluids, however, are not considered infectious. Persons with known or suspected Creutzfeld-Jakob disease, or with exposure to potential sources of iatrogenic infection, should not be considered as donors for any tissues or biologic products. Occupational transmission to health care and pathology workers is also possible. Therefore, specific preventive measures are necessary. Many questions remain regarding transmission and risk factors for Creutzfeld-Jakob syndrome, and the precautions presented here must be considered only preliminary.

From slow virus to prion: a review of transmissible spongiform encephalopathies.

Spongiform encephalopathies include seven neurodegenerative diseases: three in man (Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease and kuru) and four in animals (scrapie, mink encephalopathy, bovine spongiform encephalopathy and chronic wasting disease in deer and elks). They are all transmissible to a variety of species, and man-to-man propagation of the diseases in the form of iatrogenic transmission has been well-documented. The infectious agent is highly unusual and the pathogenesis of infection remains controversial. The term prion was introduced to describe the proteinaceous infectious agent. Purification of this agent yielded a unique sialoglycoprotein, associated with the neuronal cell membrane, which is all or part of the infectious agent. Molecular genetics revealed variations in the prion protein; these are linked to or associated with the inherited forms of spongiform encephalopathies: familial Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease. The histological triad of spongiform change, neuronal loss and astrocytosis dominate the histological picture of spongiform encephalopathies. A recent case which did not develop any of the histological hallmarks of disease, but did have genetic abnormalities typical of the disease, indicates that the true incidence of Creutzfeldt-Jakob disease may be considerably higher than previously accepted, and a combination of molecular screening and immunohistochemistry for prion protein should complement traditional neuropathology to establish the diagnosis. The descriptive term of spongiform encephalopathy may now have to be abandoned in favour of prion disease.

Prion diseases and the frame-shifting hypothesis

A group of neurological diseases, which includes scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is caused by prion agents which appear to be comprised solely of an abnormal variant of a cellular protein, PrP. Infectious agents which contain no genetic material pose a conundrum for molecular biologists. Theoretical analysis of the gene encoding PrP has suggested a possible mechanism of replication for this proteinaceous infectious agent. The putative mechanism involves frame-shifting during translation of the PrP gene. The normal and abnormal forms of PrP are predicted to have different amino acid sequences.

Genetic information and the determination of functional organization in biological systems

AbstractThe Central Dogma of molecular biology describes the one‐way flow of information from DNA to RNA to protein, but it excludes the possibility of proteinaceous infectious agents (prions) such as those implicated in certain neurological diseases like scrapie and Creutzfeldt‐Jakob disease. Rather than carrying genetic information like viruses, prions appear to carry functional information which they use to alter the meaníng of host genes during their cycle of replication. Prions represent an important counter‐example to the accepted theories of molecular genetics, in which biological specificity is determined completely by macromolecular sequence information. Criticism of the proposition that organisms can be constructed from their genes leads to the conclusion that the neo‐Darwinian interpretation of the theory of evolution is seriously deficient, especially in relation to the prebiotic phase. It is suggested that the evolutionary emergence and development of biological function, or teleopoiesis, is as important a determinant of biological evolution as the mutation and selection of genetic information.